Brown HT (formerly Chocolate Brown HT) was evaluated by JECFA in
    1977 (Annex I, Ref. 44 and 45) and a temporary ADI of 0-0.25 mg/kg
    body weight was allocated pending multigeneration reproduction/
    teratology studies and metabolic studies in several species, including
    man. The results of a teratology study were available to JECFA in 1979
    (Annex I, Ref. 50) and the Committee were informed that reproduction
    and metabolic studies had been initiated; the temporary ADI was
    extended until 1981. Further extensions of the temporary ADI were made
    in 1981 (Annex I, Ref. 56 and 57) and, following the submission of
    results of a metabolism study, again in 1982 (Annex I, Ref. 59 and

         Since the previous evaluations additional data has become
    available and is summarized and discussed in the following monograph



    Absorption, distribution and excretion

         The absorption, distribution and excretion of 14C-labelled Brown
    HT was investigated following administration of single oral doses of
    70 mg/kg body weight or 250 mg/kg body weight to male mice, or of
    50 mg/kg body weight or 250 mg/kg body weight to male and female
    rats, and male guinea pigs. In mice and rats, at both dose levels,
    substantially all of the radioactivity was excreted within 72 hours in
    the faeces (80-90%) and urine (7-16.5%); only traces of activity
    (<0.2%) appeared in expired air. In the guinea pig, faecal excretion
    appeared somewhat slower, possibly due to coprophagy, and
    approximately 90% of the dose was eliminated in 72 hours in faeces
    (74-75.5%) and urine (13-16%) with traces in expired air.

         Pretreatment of male rats with unlabelled1 Brown HT in the diet
    (0.25% = approx. 250 mg/kg/d) for 21 days prior to dosing with
    14C-labelled Brown HT (250 mg/kg per os) had no effect on the route
    of excretion or the time taken to eliminate the radioactivity.


    *    Monograph addendum

         Pregnant rats given a single oral dose of 50 mg/kg body weight or
    250 mg/kg body weight. 14C-labelled Brown HT on day 8 of pregnancy
    eliminated the radioactivity in a similar manner and at a similar rate
    to non-pregnant females.

         Following a single oral dose of 14C-labelled Brown HT of
    50 mg/kg body weight to male rats, approximately 0.05% of the
    radioactivity remained in the tissues after 72 h, mostly associated
    with the gastrointestinal tract (0.04%); the liver and kidney
    contained 0.005% and 0.006% of the activity respectively. Essentially
    similar results were obtained with male mice given a single dose of
    70 mg/kg body weight or 250 mg/kg body weight except that the levels
    of activity of the gastro-intestinal were somewhat higher (0.16%
    of the 70 mg/kg dose). Female rats given a single oral dose of
    14C-labelled Brown HT of 250 mg/kg body weight retained approximately
    0.25% of the administered dose in the tissues after 72 hours, mainly
    in the gastro-intestinal tract (0.17%); the liver and kidney contained
    0.04% and 0.014% of the activity, respectively.

         In order to investigate whether prolonged exposure to Brown HT
    increased the body burden of the colour or metabolites, male rats were
    given 14-labelled Brown HT by gavage at a dose level of 250 mg/kg per
    day for 21 days. The animals were killed 24 hours after the last dose
    and radioactivity measured in liver, kidney, spleen, stomach, small
    intestine, caecum and lymph nodes (neck, axilliary, and mesenteric).
    The radioactivity in these organs, expressed as a percentage of the
    total dose, was low and associated mainly with the gastro-intestinal
    tract (0.275%); the radioactivity in liver, kidney and spleen was
    0.015%, 0.009% and 0.004% of the total dose, respectively. The
    mesenteric lymph nodes contained 0.001% of the total radioactivity

         Tissue levels of radioactivity were measured in pregnant and non-
    pregnant female rats 96 hours after administration of a single oral
    dose of 250 mg/kg body weight of 14C-labelled Brown HT; the pregnant
    rats were dosed on day 8 of pregnancy. In both pregnant and non-
    pregnant animals, approximately 0.30% of the dose remained in the
    tissues examined viz: liver, kidney, spleen, heart, lung, ovaries,
    brain, stomach, intestine and caecum. In non-pregnant animals, most of
    the residual activity was associated with the gastro-intestinal tract,
    liver, kidney and brain while in the pregnant animals the highest
    levels were found in kidney, heart, and lung. In the pregnant animals
    a further 0.23% of the dose was found in fetuses.

         The excretion of radioactivity in the bile of three male rats was
    measured for up to 7 hours after administration of a single oral dose
    of 250 mg/kg body weight of 14C-labelled Brown HT. Less than 0.6% of
    the dose was eliminated by this route, with both the rate of bile flow
    and rate of excretion of radioactivity approximately constant over
    this time period.

         The intestinal absorption of 14C-labelled Brown HT was
    investigated using isolated loops of small intestine from mouse, rat
    and guinea pig in vivo. No significant absorption of radioactivity
    was detected over a one-hour period in any of the species investigated
    for concentrations of Brown HT from 50 to 5.000 mg/l (Gaunt, Phillips
    & Hendis, 1981).

         A second study was performed in rats to clarify whether repeated
    administration of Brown HT led to tissue specific or irreversible
    accumulation of the colour or its metabolites. A single oral dose of
    250 mg 14C-labelled Brown HT/kg body weight was administered to
    unpretreated male rats and to rats which had been given a similar
    daily dose of unlabelled or 14C-labelled Brown HT for 21 days.
    Urinary and faecal excretion, and tissue levels of radioactivity were
    monitored 24, 48, 72 and 168 hours after the last dose; the tissues
    examined included liver, kidney, spleen, heart, lung, brain, testes,
    stomach, small intestine, caecum, large intestine, lymph nodes (neck,
    axilliary and mesenteric) and blood.

         Following the administration of 14C-labelled Brown HT to
    unpretreated rats, the majority of the dose was excreted rapidly in
    urine and faeces, mainly within 48 hours. Substantial amounts of
    radioactivity were found in the tissues 24 hours after dosing, with
    the highest concentrations in the gastro-intestinal tract, lymph nodes
    and kidney. By 168 hours after dosing the concentration of
    radioactivity was similar in all tissues (less than 0.001% of the
    dose/g tissue) except the kidneys (0.009%) and mesenteric lymph nodes
    (0.003%/g); the concentration of radioactivity remained similar from
    48 hours to 168 hours after dosing.

         Similar results were seen in animals pretreated for 21 days with
    unlabelled Brown HT prior to the single oral dose of 14C-labelled
    colour. There was no significant difference in the proportion of the
    administered dose excreted in the urine compared to non-pretreated
    animals and the tissue concentrations of radioactivity at 168 hours
    were similar. However, at the earlier times, the mean concentration of
    radioactivity in the liver, kidney and lymph nodes was somewhat
    greater than in the non-pretreated animals, and the concentration in
    the kidney fell from 0.02% dose/g tissue at 48 hour to 0.0075% dose/g
    tissue after 168 hours.

         The distribution of radioactivity in the tissues of animals
    pretreated for 21 days with 14C-labelled Brown HT was similar to that
    in the other two groups although the amount and concentration of
    radioactivity present in most organs was greater at each of the times

         The authors concluded that either Brown HT and/or metabolites
    accumulated in most tissues of male rats during repeated daily
    administration of 250 mg/kg body weight per os but that only in the

    kidney and mesenteric lymph nodes was the accumulation tissue
    specific.  In other organs, the accumulated colouring was cleared
    rapidly on cessation of treatment (Phillips, Mendis & Gaunt, 1982).


         24 hours urine and faecal samples from mice, rats and guinea pigs
    in the first excretion study described above were examined by thin-
    layer chromatography. Two metabolites were detected in the urine; the
    major component (65-90% of the urinary radioactivity) was identified
    as naphthionic acid and the minor component was not identified.

         The faecal extracts contained small amounts of unchanged Brown HT
    (1.5-6.5% of the dose) together with naphthionic acid and two
    unidentified metabolites (Gaunt, Phillips & Mendis, 1981).


    Special studies on reproduction

         A multigeneration study was performed in rats, in which males and
    females received Brown HT at dietary concentrations designed to
    provide daily intakes of 0, 50, 250 or 500 mg/kg body weight for three
    successive generations.  The F2 generation comprised two consecutive
    litters, designated F2a and F2b. A teratogenicity study was carried
    out with 12 females per test group (24 controls) of the Fo, F1 and
    F2b generations.  A gross post-mortem examination was carried out on
    excess weanlings of the F1, F2a and F2b generations and a full
    post-mortem examination on breeding adults of the Fo, F1, F2a and
    F2b generations after their litters had been weaned. In addition, a
    full post-mortem was performed on 1 male and 1 female from each litter
    from the F2a generation 33 + 2 days post partum, on 1 male and 1
    female from each litter of the F2b generation 70 + 2 days post partum
    and on 3 males and 3 females of each litter (where possible) of the
    F3 generation 70 + 2 days post partum.

         In the full post-mortem examination, adrenals, brain, caecum
    (with and without contents), gonads, heart, kidneys, liver, spleen,
    stomach and thyroid gland were removed and weighed (thyroid was not
    weighed at autopsy of the F2b and F3 generations); other tissues
    sampled but not weighed were aorta, colon, eye, harderian gland,
    lungs, cervical and mesenteric lymph nodes, mammary tissue,
    oesophagus, pancreas, rectum, salivary glands, skeletal muscle, skin,
    small intestine, spinal cord, thymus, trachea, urinary bladder, vena
    cava, seminal vesicles and epididymis or uterus and vagina.
    Histopathological investigations were carried out on the tissues taken
    at autopsy from control and high dose (500 mg/kg body weight) groups
    of the F3 generation after staining with haematoxylin and eosin.

         At autopsy, brown colouring was evident in the lining of the
    gastro-intestinal tract and, more particularly, in the lumph nodes of
    some treated animals. The intensity of the colour appeared to be
    related to the level and duration of treatment, however, there were no
    histological changes in these tissues that could be attributed to
    treatment and the body weights, food and water consumption and general
    condition of the animals were unaffected.

         The post-mortem examinations and organ weights of animals that
    had received up to 250 mg Brown HT/kg body weight per day did not
    reveal any adverse effects related to treatment. At the highest dose
    level (500 mg Brown HT/kg/d) caecal enlargement was a frequent finding
    in adult females and the kidney weights were consistently higher than
    controls; there was no histological evidence of toxic effects in these

         The reproductive performance of the animals was unaffected by
    treatment. In the teratology studies examination of the uterine
    contents of pregnant females of the Fo, F1 and F2 generations did
    not reveal any treatment related differences in the number of ova that
    implanted, the number of implantations that resulted in viable fetuses
    or in the weight and appearance of the fetuses. Slight differences in
    degree of skeletal ossification were seen in fetuses from F2
    generation dams but were considered only minor variation of normal.

         There were no adverse effects of treatment on the post-natal
    development of the offspring from females allowed to litter, as judged
    by survival, body weight and achievement of developmental milestones
    (eye opening, unfolding of the pinna, tooth eruption, hair growth,
    righting reflex, startle reflex, clinging ability).

         With respect to reproduction, the no-effect-level was considered
    to be 500 mg Brown HT/kg body weight per day; however, on the basis of
    the changes in kidney weight, it was concluded that the no-untoward-
    effect level was 250 mg Brown HT/kg body weight per day (Brantom et
    al., 1981).

    Special studies on teratogenicity and embryotoxicity

         Groups of 30 pregnant Wistar rats were given daily oral doses of
    Brown HT of 0, 250, 500 or 1000 mg/kg body weight from day 0 to day 19
    of pregnancy. The animals were weighed on alternate days from day 0 to
    20 of pregnancy when they were killed by cervical dislocation. At
    autopsy, the numbers of corpora lutea and implantation sites were
    recorded together with the numbers and positions of the sites of dead,
    living or resorbed fetuses. Live fetuses were weighed, examined for
    gross abnormalities and preserved in alcohol or Bouins solution. Those
    preserved in alcohol were examined for skeletal abnormalities after
    alizarin staining and those preserved in Bouins solution were examined
    for soft tissue abnormalities using the Wilson technique.

         There was no effect of treatment on implantation, pre- and post-
    implantation losses, litter weight, fetal weight or sex ration. No
    treatment-related abnormalities were found in skeletal preparations or
    gross sections of the fetuses. It was concluded, therefore, that doses
    of up to 1000 mg Brown HT/kg body weight per day did not exert any
    fetotoxic or teratogenic effect.

    Special studies on pigment deposition of mesenteric lymph nodes and

         In view of the observations in metabolism studies, earlier
    toxicological studies and the multigeneration study (see above) that
    pigment deposition occurred in mesenteric lymph nodes and possibly
    in kidneys, which were enlarged, an independent evaluation of
    histopathologcal material from the F3 generation of the
    multigeneration study was performed. Sections from 2 male and 2 female
    controls and from 5 top dose (500 mg/kg body weight) males and 5 top
    dose females were examined as follows: thyroid, heart, liver, kidney,
    voluntary muscle, caecum, thymus, mesenteric lymph node and cervical
    lymph node. Sections of mesenteric lymph node only from a further 10
    top dose males and 5 top dose females were also examined.

         No evidence of pigment deposition was found in any of the tissues
    examined.  Specifically, there was no pigment deposition in follicular
    cells of the thymus, Kupffer cells of the liver, proximal convoluted
    tubules or other sites in the kidney nor was there any evidence of
    pigment deposition in cardiac or voluntary muscle or in the caecal
    wall. Finally, in the mesenteric lymph nodes, there were no pigment-
    laden macrophages in the sinuses and no other evidence of pigment

         It was concludeed that exposure over 3 generations to this dose
    of Brown HT gave rise to no accumulation of pigment in a form which
    survived normal tissue processing.  No histopathological changes were
    seen in any of the tissues examined and it was not possible to
    distinguish between the mesenteric lymph nodes of treated and control
    rats, with no evidence of stimulation or atrophy of lymphoid tissue
    (Roe, 1983).


         The multigeneration/reproduction and teratology studies indicate
    that Brown HT is without effect on reproduction at daily doses up to
    500 mg/kg body weight over 3 generations. The caecal enlargement and
    increase in kidney weight observed at the highest dose levels was not
    associated with any histopathological changes and appeared
    asymptomatic. The pigment observed in some tissues in these studies
    after administration of high doses of Brown HT did not survive normal
    tissue processing and no accompanying histopathological changes could
    be detected in mesenteric lymph nodes or kidney.

         Earlier long-term studies (Annex Ref. TRS 617) in two rodent
    species indicated that Brown HT is not carcinogenic in these species
    and a no-effect-level of 0.1% in the diet was established in the


    Level causing no toxicological effect

         Mouse:    0.1% (= 1000 ppm) in the diet, equivalent to
                   150 mg/kg bw.

    Estimate of acceptable daily intake for man

         0-1.5 mg/kg bw.


    BUTLER, W.H., CONNING, D.M. (1981) Multigeneration toxicity
    study with Brown HT in rats. Unpublished BIBRA Report No. 294/1/81
    submitted to WHO.

    GAUNT, I.F., PHILLIPS, J.C., & MENDIS, D.  (1981) The metabolic
    disposition of 14C-labelled Brown HT in the rat, mouse and guinea
    pig. Unpublished BIBRA Research Report No. 1/1981 submitted to WHO.

    PHILLIPS, J.C., MENDIS, D., & GAUNT, I.F. (1982) The absorption,
    tissue distribution and excretion in the rat of Brown HT following
    single oral and sub-acute oral treatment. Unpublished BIBRA Research
    Report No. 2/1982 submitted to WHO.

    ROE, F.J.C. (1983) Brown HT toxicological assessment with special
    reference to pigment deposition in mesenteric lymph nodes and kidneys.
    Unpublished report from the EEC Colours Group submitted to WHO.

    See Also:
       Toxicological Abbreviations
       BROWN HT (JECFA Evaluation)