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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

    WORLD HEALTH ORGANIZATION



    SUMMARY OF TOXICOLOGICAL DATA OF CERTAIN FOOD ADDITIVES



    WHO FOOD ADDITIVES SERIES NO. 12






    The data contained in this document were examined by the
    Joint FAO/WHO Expert Committee on Food Additives*
    Geneva, 18-27 April 1977




    Food and Agriculture Organization of the United Nations
    World Health Organization



    * Twenty-first Report of the Joint FAO/WHO Expert Committee on Food
    Additives, Geneva, 1977, WHO Technical Report Series No. 617

    CARAMEL COLOUR (AMMONIA AND AMMONIA-SULFITE PROCESS)

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    BIOLOGICAL DATA

    BIOCHEMICAL ASPECTS

         Caramel refers to a large number of poorly defined and complex
    products formed from various carbohydrates generally by heating with
    any of a wide range of acids, bases and salts, under varying
    conditions of temperature and pressure. It might be argued that
    caramel can be considered as a natural constituent of the diet as it
    can be formed when certain foods are cooked or when sucrose is heated.
    Toxicological discrimination is unwarranted between such caramel and
    caramels produced commercially from food-grade carbohydrates, with the
    exception of caramels prepared by processes using ammonia or ammonium
    salts.

         The production of violent hysteria and convulsions in cattle and
    sheep fed ammoniated sugar-containing feed supplements (nitrogen
    content 4-6%), at 6-25% of their rations led to the discovery of the
    presence of about 20% of pyrazines and 10% of imidazoles in these
    ammonium-treated molasses. 4-methylimidazole has been shown to be the
    most likely toxic component being a convulsant to rabbits, mice and
    chicks at oral doses of 360 mg/kg body weight (Nishie et al., 1969).
    The pyrazines, on the other hand, are mild CNS depressants and weak
    anticonvulsants (Heyns, 1970). Analysis of food grade caramel colours,
    however, showed that only 0.002-0.02% of 4-methylimidazole is present
    in commercial products (Heyns, 1970). Commercial caramel colours of
    undefined origin contain 50-500 ppm 4-methylimidazole (Heyns, 1971)
    while other examinations have shown ranges of 100-700 ppm (Battelle
    Memorial Institute, 1971). It has been shown that the yields of
    imidazole compounds increased linearly with the increment of molar
    ratio of ammonia to glucose (Komoto, 1962). Further analyses for
    4-methylimidazole were being carried out on a large variety of caramel
    colours (Nishie et al., 1970). Generally caramel colours contain 50%
    digestible carbohydrate, 25% non-digestible carbohydrate and 25% of
    melanoidins also found in roasted coffee, broiled meats and baked
    cereal products.

         In groups of two to four rats, the absorption of the colour-
    giving components of caramel was determined by faecal extraction.
    Recoveries varied widely for the 10 or 20% caramel solutions examined
    despite pre-treatment for 100 days before testing. About one-third of
    the colour-giving components appeared to be absorbed but no
    conclusions could be drawn regarding the absorption of colourless
    components (Haldi and Wynn, 1951).

    TOXICOLOGICAL STUDIES

    Special studies on reproduction

         Fifteen male and 15 female rats were given 0 or 10% ammonia-
    sulfite caramel solution as their sole fluid source until day 100 and
    were then mated. The F1-generation (25 males and 25 females) was
    weaned and given again 0 or 10% caramel solution until day 100. It
    shows no adverse effects as regards number of litters born and number
    of pups/litter. No influence on haematology, growth, food consumption,
    gross and histopathology of the F1-generation at an age of 100 days
    was found (Haldi and Wynn, 1951). Six different ammonia-sulfite
    caramels (three single strength (SS) and three double strength (DS)
    products) each containing a different level of 4-methylimidazole
    (between 200 and 850 ppm) were tested in a reproduction study with
    rats. Two control diets were used viz. an unmodified stock diet and
    the stock diet supplemented with starch and cellulose. The various
    test diets enable to examine the effects of 5, 10 and 15% of a single
    strength caramel, and of 2.4 and 6% of a double strength caramel and
    of 10% single strength and 4% double strength caramels each with three
    different contents of 4-methylimidazole (see Table 1).

         Twelve groups of 30 rats (a Wistar strain), 10 males and 20
    females per group were used. At week 12 all rats were mated in groups
    of five males and ten females to produce one litter.

         At weaning age of the young 40 males and 40 females were selected
    from as many different litters as possible of each caramel group and
    continued on the same diet as their parents. Ten males and ten females
    of each group are intended to be sacrificed after one year (see short-
    term studies), the remaining 30 males and 30 females of each group are
    intended for completing a two-year feeding period (see long-term
    studies). After weaning the first litters, the mothers were killed to
    count the implantation sites.

         No influence on growth of the parents was noted. After the
    breeding no adverse effects on the number of young per litter, average
    weight and growth of the young, mortality (except in the group with
    10% SS + 600 ppm methylimidazol, no significant increase was found),
    ratio males/females and number of implantation sites was found. No
    teratogenic effects were found (Til and Spanjers, 1973).

        TABLE 1.  COMPOSITION OF THE TEST DIET (IN g)
                                                                                                        

    Group               1      2      3      4      5      6      7      8      9      10     11     12
                                                                                                        

    Caramel in %        5      10     15     10     10     2      4      6      4      4      0      0
                                                                                                        

    stock diet          100    100    100    100    100    100    100    100    100    100    100    100

    wheat starch        3.8    1.9    -      1.9    1.9    4.9    4.1    3.4    4.1    4.1    5.8    -

    cellulose           4.2    2.2    -      2.2    2.2    5.4    4.6    3.8    4.6    4.6    6.2    -

    SSa caramel
    (ammonia-sulfite
    process) + 202b     5.6    11.5   17.6

    SS caramel
    (ammonia-sulfite
    process) + 400                           11.5

    SS caramel
    (ammonia-sulfite
    process) + 600                                  11.5

    DSc caramel
    (ammonia-sulfite
    process) + 350                                         2.3    4.5    6.8

    DS caramel
    (ammonia-sulfite
    process) + 639                                                              4.5
                                                                                                        

    TABLE 1.  COMPOSITION OF THE TEST DIET (IN g) (con't)
                                                                                                        

    Group               1      2      3      4      5      6      7      8      9      10     11     12
                                                                                                        

    Caramel in %        5      10     15     10     10     2      4      6      4      4      0      0
                                                                                                        

    DS caramel
    (ammonia-sulfite
    process) + 852                                                                     4.5
                                                                                                        

    a  SS = Single strength.         b  Quantity of methylimidazole in ppm.
    c  DS = Double strength.
    
    Special studies on teratogenicity

         Groups of 19-22 pregnant female mice (strain CD-1) were used in
    this experiment. The mice reeeived by oral intubation 0.16, 74.3, 345
    and 1600 mg caramel per kg body weight for 10 consecutive days. The
    caramel type was described as "beverage" (ammonia-sulfite process).
    The administration was started on day six continuing daily through day
    15 of gestation. Appearance, behaviour, food consumption and growth
    were studied. On day 17 all dams were subjected to Caesarcan section
    and the number of implantation sites, resorption sites, liver, dead
    foetuses and body weight of the pups were recorded. All foetuses were
    examined grossly for external congenital abnormalities, one-third of
    the foetuses of each litter underwent visceral examination (Wilson
    technique), the remaining two-thirds were stained with alizarin for
    skeletal defects.

         The number of abnormalities seen in either soft or skeletal
    tissues of the test groups did not differ from the number occurring
    spontaneously in the sham-treated controls (Morgareidge, 1974a).

         Virgin adult female rats (strain Wistar) were given by oral
    intubation 0, 16, 74.3, 345 and 1600 mg caramel/kg body weight for
    10 consecutive days. The caramel types was described as "beverage"
    (ammonia-sulfite process).

         The administration was started on day six and continuing daily
    through day 15 of gestation. On day 20 the animals were subjected to
    Caesarcan section. The same parameters as studied in the mouse
    experiment were also studied in the rat.

         No clearly discernible effect on nidation or on maternal or
    foetal survival was found. The number of abnormalities found in the
    test groups did not differ from that occurring spontaneously in the
    sham-treated controls (Morgareidge, 1974a).

         Virgin adult Dutch-belted female rabbits were given 0, 16, 74.3,
    345 and 1600 mg caramel/kg body weight by oral intubation. The caramel
    type was described as "beverage" (ammonia-sulfite process). The
    administration started on day six continuing daily through day 18. On
    day 29 all doses were subjected to Caesarean sections and all the same
    parameters as mentioned under mouse and rat were studied.

         No clearly discernible effect on nidation or on maternal or
    foetal survival were seen. The abnormalities seen in either soft or
    skeletal tissues of the test groups did not differ from that
    occurrency spontaneously in the sham-treated controls (Morgareidge,
    1974b).

         A study in mice, rats and rabbits was carried out as described
    above for mouse, rat and rabbit separately. The caramel was described
    as "bakers and confectionary caramel". Caramel was given by gavage in
    doses of 0, 16, 74.3, 345 and 1600 mg/kg body weight during the above
    stated periods of gestation. No abnormalities were seen (Morgareidge,
    1974a and b).

    Special studies on mutagenicity

         Caramel (not described what was tested) was evaluated for genetic
    activity in a series of in vitro microbial assays with and without
    metabolic activity. Salmonella typhimurium (strain TA-1535, TA-1537
    and TA-1538) and Saccharomyces cerevisiae was used. This caramel was
    not genetically active under the test conditions employed in this
    study (Brusick, 1974).

    Special studies on metabolites

         Male albino mice (20-25 g) were used to determine the median
    convulsive dose (CD50) and the median lethal dose (LD50) of a few
    imidazoles. The results are given in Table 4.

    TABLE 4.  CONVULSANT AND LETHAL EFFECTS OF IMIDAZOLES
                                                                        

                            CD50 + SE                LD50 + SE
                           in mg/kg bw              in mg/kg bw
                                                                        

                        intrap.    oral          intrap.    oral
                                                                        

    4-methylimidazole   155  5    360  18      165  3    370  15
    1-methylimidazole   380  8.2  1 400  79    380  8.2  1 400  79
    2-methylimidazole   500  12   1 300  70    480  18   1 400  114
    imidazole           560  34   1 880  45    610  7.4  1 880  45
                                                                        

         All the imidazoles tested produced varying degrees of tremor,
    running, restlessness, siaborrhea, Straub tail, opisthotonus and tonic
    extensor seizure that ended in death (Nishie et al., 1970).

         The CD50 and the LD50 of 4-methylimidazole in one-day-old chicks
    was respectively the intraperitoneal CD50 ( SE) was 174  10 mg and
    the LD50 210  15 mg/kg body weight. Per orally the CD50 was 580  30
    mg and the LD50 was 599  50 mg/kg body weight. Doses of 100 mg/kg
    intraperitoneally caused tremors, peeping and spreading of the wings.
    Doses over 150 mg/kg body weight intraperitoneally caused
    opisthotonus, prostration with clonic leg movements and terminal tonic
    extensor seizure (Nishie et al., 1970).

    Acute toxicity

                                                                   

                             LD50                Reference
    Animal    Route     mg/kg body weight
                                                                   

    Rat       Oral      >2.3 ml equiv. 1 900     Foote et al., 1958
              Oral      > 25 ml equiv. 17 500    Chacharonis, 1960
              Oral      >30 ml equiv. 20 400     Chacharonis, 1963
                                                                   

         No abnormalities were detected after observation of animals for
    14 days following administration of 12 different caramel colour
    products mostly based on ammonia or ammonium sulfate catalysts (Foote
    et al., 1958; Chacharonis, 1960; Chacharonis, 1963). A single dose of
    up to 10 g/kg body weight in mice and 15 g/kg in rabbits of caramels
    produced by the ammonia catalyser closed pan process or sodium
    hydroxide process did not cause convulsions or other signs of distress
    (Sharratt, 1971).

    Short-term studies

    Mouse

         In a four to six week study, albino Swiss mice (10 males and 10
    females per group) were given caramel (ammonia process) containing
    830 ppm 4-methylimidazole in the following dose levels 0, 1, 2, 4, 8
    and 16% in the diet. No influence on appearance, behaviour and food
    intake were noticed. The growth was especially in the group with 16%
    decreased in the third and fourth week. The faeces of the animals
    especially with the higher dose-levels was soft, tarry in appearance
    and assumed a sticky, pasty consistency and poorly formed.

         In the males with 16% caramel an increase of neutrophilic
    leucocytes and a decrease in lymphocytes were noticed. The mean
    relative weight of the caeca (full weight and empty weight) was partly
    or clearly increased in the 4, 8 and 16%. About the histopathological
    study only is mentioned that there were no remarkable findings.
    Histopathological examinations were not fully reported (Procter,
    1976).

    Rat

         This study is a part of the experiment described under
    reproduction studies. It was further the outcome of the interim
    sacrifice of one-fourth of the animals utilized in the long-term study
    described below.

         The rats (10 males and 10 females in each group) were selected
    from the first litter of parents that were given diets containing
    different caramels (anmaonia-sulfite process) (three single strength
    (SS) and three double strength (DS), containing 200-850 ppm of
    4-methylimidazole) in different dose levels (5, 10 and 15% (SS) and
    2.4 and 6% (DS); furthermore 3 (ammonia-sulfite process) SS-caramels
    with different methylimida-zole contents were tested in 10% and 3
    (ammonia-sulfite process) DS-caramels with different methylimidazole
    content were tested in 4% in the diet) (see Table 2). Only group 12
    was not included in this experiment. The animals were sacrificed after
    a feeding period of one year.

         In this study on behaviour, growth, food intake, mortality, liver
    and kidney function tests, urine composition, organ weights, no
    adverse effects were found. Haematological indices showed a slight
    dose-related decrease in total leucocyte counts in females fed
    SS-earamel containing 202 ppm of methylimidazole. In males no effect
    was noticed. In a subsequent experiment in which the same SS-caramel
    was fed to female rats at levels as high as 15 and 30% no indications
    of decreased leucocyte counts were observed after 4, 8, 12 or 16
    weeks.

         The only finding which is attributed to the feeding of the
    caramels consisted of increased accumulations of a yellow-brown
    pigment and pigment laden macrophages in the megenteric lymph nodes of
    males and females in all caramel groups. Inflammatory or degenerative
    changes of the lymphoid tissue was not found (Sinkeldam et al., 1975a
    and b).

         Three groups of 20 females (Wistar) received the stock diet to
    which 0, 10 and 20% of ammonia-sulfite process, SS-caramel containing
    202 ppm 4-methylimidazole was added. During the second week of the
    experiment these levels were increased to 15 and 25% and in week seven
    increased respectively to 25 and 30%. Throughout 16 weeks the diets
    were administered and followed by a four-week recovery period, without
    the caramel.

         The food consumption and growth of the test animals were
    comparable with the controls. Leucocyte counts, collected after 4, 8,
    12 and 16 weeks did not show statistically significant differences
    among the groups. The relative weights of the caecum, both filled and
    empty were distinctly increased already after four weeks of caramel
    feeding. After a recovery period of four weeks the increases had
    disappeared. The relative weights of the thymus was not affected.

         Gross examination at autopsy after 16 weeks revealed a dose-
    related, brown-greenish discoloration of the mesenteric lymph nodes in
    all test animals of the highest dose level. After the recovery period
    of four weeks the colour change was less, but still visible.

    Microscopically the lymph nodes of the test rats showed pigment
    accumulation which was not noticeably diminished after withdrawal of
    the caramel for four weeks. These results failed to confirm the
    decreased leucocyte count which was observed in females fed 5 up to
    15% SS-caramel 202 in the one year feeding study (Sinkeldam et al.,
    1975a).

         Seven groups of 20 female rats (Wistar) were fed 0, 15 and 30% of
    three types of caramel containing 1750 of 4-methylimidazole. The
    caramels were manufactured by using catalysts ammonia. The
    administration of the caramel was for eight weeks followed by a four-
    week recovery period.

         The diets containing caramel caused dose-related decrease in body
    weights and food efficiency caused diarrhoea. Leucocyte counts after
    4, 8, 10 and 12 weeks did not show differences with the controls. The
    relative weights of the caecum, both filled and empty were
    considerably increased already after four weeks.

         After a recovery period of four weeks the increases had
    disappeared. Gross examination at autopsy after eight weeks revealed a
    slight, dose-related brown discoloration of the mesenteric lymph
    nodes, in a few animals of each test group. After recovery periods of
    two or four weeks the discoloration was less intensive, but still
    visible. Microscopically the lymph nodes of the test rats showed
    accumulation of pigment-laden macrophages, which was not noticeably
    diminished after withdrawal of the caramel for two or four weeks
    (Sinkeldam and Van der Heyden, 1975/1976).

         Six groups of 15 rats (CFE-strain) of each sex were given diets
    for 10 weeks containing 4, 8 or 16% caramel (ammonia-process)
    (produced by either an "open" or a "closed" pan ammonia process) (no
    indications are given about the content of 4-methylimidazoles in these
    caramels). A group of 25 rats of each sex served as controls. There
    was a decrease in body weight gain at all dietary levels of both
    caramel types. There were reduced haemoglobin concentrations in the
    highest dietary level, while in the lower levels this effect was less
    clear. After 13 weeks in the males of all the dose levels the
    Hb-content was significantly decreased. In the females only in the 8
    and 16% levels. In certain cases, but less consistent, the total
    number of red blood cells was lowered. The total number of leucocytes
    was significantly decreased and a lymphocytopenia was present at all
    levels in association with lower weight of the thymus and the spleen
    in the 8 and 16% level. The caecum weights were increased compared
    with the controls. Increased relative liver and kidney weights
    suggested an effect on these organs. Changes in the weights of other
    organs were considered to be related to the differences of body weight
    between the groups. The volumes of urine excreted during a six-hour
    period without water or in the two-hour period after a water load were

    lower than the control values. The latter differences were accompanied
    by higher values for the specific gravity of the urine. The
    histopathological study did not reveal treatment-related changes (no
    details are given) (Gaunt et al., 1975).

         Sixteen groups of rats were given for 10 weeks different dose-
    levels of three caramel types. Weanling rats (strain Wistar) per
    group, 15 males and 15 females, except in the control group in which
    60 animals of each sex and in the three groups with the highest dose
    level of caramel, in which 10 animals of each sex were used.

         The caramels that were tested were single strength ammonium
    sulfite (MS), double strength ammonium sulfite (DS) and caramel
    ammonia-process in dose levels of 0.5, 1.0, 2.0, 4.0 and 16.0% in the
    diet (no data on the presence of 4-methylimidazole are given. Food
    intake, growth was estimated and haematological examination and
    histopathological studies were carried out. Especially lymph nodes,
    thymus, spleen and caecum were studied for distribution of pigment.

         Another four groups of 10 rats were given basal diet or diet
    containing 16% of one of the caramels for 10 weeks. At the end of this
    experiment all the rats received basal diets. Five rats of each sex
    were killed after seven days and the remainder after 28 days of
    feeding basal diet (recovery experiment).

         In the group with 16% DS and 2% ammonia process caramel decreased
    body weight gain was found. The total leucocyte count showed in the
    males in the groups with 2, 4 and 16% ammonia process caramel and less
    clear in 4 and 16% DS caramel was decreased. In the females this was
    only in the 16% ammonia process caramel. However, the lymphocyte/
    neutrophil ratio was significantly decreased in all dose levels
    ammonia process caramel. The relative liver weight was increased in 2%
    ammonia and 2% DS caramel and higher levels. Reduced spleen weight was
    found in 16% ammonia process caramel. Increased relative kidney weight
    was already seen in the 2% ammonia process caramel, 2% DS and 16%
    SS-caramel group. Increased caecal weight was only seen in 16% of all
    the three caramel types. The pigment in the lymph nodes was seen in
    16% DS caramel. Microscopically pigments were found in mesenteric,
    cervical and axillary lymph nodes in the males in the groups with 4
    and 16% SS, 4 and 16% DS caramel and only in the mesenteric lymph
    nodes of the 16% ammonia process caramel. In the females this was only
    found in the 16% DS caramel and 16% ammonia-process caramel.

         After the treatment was stopped the white cell counts, cell
    ratios and the total number of lymphocytes rapidly returned to normal
    values. This recovery was complete in both sexes by seven days. The
    increased relative liver weights did not return completely to normal
    during the recovery phase, the kidneys and spleen weights recovered
    partly to normal. The caecal weights changed fast to normel in seven
    days (BIBRA report, 1977).

         In a 10-week feeding study 17 groups of 15 male and 15 female
    rats (strain Sprague-Dawley) were given various caramels in different
    dose levels. The caramels were an ammonia process caramel (containing
    830 ppm 4-methylimidazole), single strength ammonia sulfite caramel
    (SS) (containing 140 ppm 4-methylimidazole) and a double strength
    ammonia sulfite caramel (DS) (containing 340 ppm 4-methylimidazole).
    The dose levels that were fed were 1.25, 2.5, 5, 10 and 15% ammonia
    process caramel and SS or 0.5, 1, 2, 4 and 6% DS caramel. Furthermore
    two control groups were used.

         In the animals with ammonia process caramel in levels of 5% and
    higher the faeces became within two weeks soft, the water content of
    the faeces was higher. This was also found in the animals with 15%
    SS caramel. There was no clear profound effect on the increase of body
    weight, except the 15% ammonia process caramel group, in this group
    was the clearest effect.

         From the blood studies it was clear that the ammonia process
    caramel caused significant reduction in lymphocyte count and a
    coincident increase in the numbers of segmented neutrophils. This
    effect was even found in the 1.25% level (but no clear dose-relation).
    No macroscopic evidence of abnormal pigmentation of the mesenteric
    lymph nodes were found. Increase in caecal size (empty weight) was
    consistently evident in animals of both sexes fed ammonia process
    caramel at the 5, 10 and 15% level. For the groups of animals with
    caramel SS and DS this was in general not the case.

         The histopathological study did not reveal changes in
    the structure of the ileal or caecal mucosae nor in the
    reticuloendothelial components of the central or peripheral systems.
    No abnormal pigmentation of the lymph nodes was found (Procter et al.,
    1976).

         A 10-week study with 18 groups of 10 male and 10 female rats
    (strain Wistar) was carried out with three different ammonia caramels
    viz. ammonium sulfite caramel single strength (SS), ammonium sulfite
    caramel double strength (DS) and ammonia process caramel. No data on
    the content of 4-methylimidazole were given. The dose levels were
    0 (control), 1.25, 2.5, 5, 10 and 15% SS caramel or ammonia process
    caramel or 0 (control), 0.5, 1, 2, 4 or 6% DS caramel. All caramels
    caused loose stools at the highest dose level, most marked with the
    ammonia process caramel (in the lower dose levels it was mentioned).
    Body weights were slightly decreased only in the group fed 15% ammonia
    process caramel, in both sexes. Leucocyte (lymphocyte) counts were
    decreased in males and females fed 15% ammonia process caramel in all
    dose levels (in the lower dose levels only in the females). The
    relative weight of the caecum (both filled and empty) was distinctly
    increased by feeding ammonia process caramel. With SS and DS caramel

    only slight indications of caecum enlargement were observed.  Minimal
    amounts of pigment were observed in mesenteric lymph nodes of several
    rats in the rats with 1.25% of ammonia process caramel 2.5 and 1% of
    SS and DS caramel respectively and the higher dose levels.

         From this experiment it appeared that the amonia process caramel
    was distinctly more active than the two other caramels in regard to
    growth depression, enlargement of the caecum and decrease in leucocyte
    counts (Sinkeldam and Van der Heyden, 1976a)

         Groups of five male and five female rats were given 1 ml/kg body
    weight of concentrated caramel colour for 21 days. Some diarrhoea was
    induced in all animals but no other abnormalities were noted. Gross
    and histopathology revealed no significant changes due to
    administration of the test compound (Foote et al., 1958).              

         Groups of five rats received either 10 or 20% caramel solution
    equivalent to about 10 or 20 g/kg body weight as sole source of fluid
    for 127 days. Only dark faeces and very mild diarrhoea were noted. No
    adverse effects were noted regarding general health, body weight, food
    and fluid consumption, haematology, gross and histopathology (Haldi
    and Wynn, 1951).

         Six groups of five male and five female weanling rats received 0
    or 10% caramel solution as their sole fluid source for 100, 200 or 300
    days respectively. No adverse effects were noted regarding growth,
    food and fluid intake, haematology, gross and histopathology (Haldi
    and Wynn, 1951).

         Groups of 16 male and 16 female rats received either 0 or 10%
    caramel solution for 100 days and groups of five rats received 20%
    caramel solution for 100 days. At the lower test level there were no
    observable abnormalities as regards growth, food consumption,
    haemetology, gross and histopathology. Only growth and haematology
    were examined at the higher test level (Haldi and Wynn, 1951).

         Three groups of 20 male and female rats received either 0 or
    11-14 g/kg body weight of caramel solutions for 100 days. Growth and
    food intake did not differ significantly between test and control
    animals. Gross and histopathology showed no abnormal findings related
    to administration of the test compound (Haldi, 1958).

         Four groups of 10 male and 10 female rats received 0, 0.1, 1.0
    and 10% of caramel colour in their diet for 12 weeks. No adverse
    effects were noted on growth, food consumption, urinalysis,
    haematology, gross and histopathology related to administration of the
    caramel colour (Prier, 1960).

         Groups of 10 male and 10 female rats received 0, 5, or 10 g/kg
    caramel colour in their diet for three months. Weight gain was normal
    in all groups. Food consumption, haematology and urinalysis were
    comparable. Gross and histopathology showed no test-related adverse
    findings (Chacharonis, 1960).

         Four groups of 10 male and 10 female rats received 0, 5, 10 and
    20% of two different caramel colours in their diet for 90 days. In
    addition, a paired feeding study involving five male rats in two
    groups was run for 23 days with one sample at the 20% level, and there
    was no difference in the rate of growth. The only effects attributable
    to treatment were a mild depression in growth of male rats at the 10
    and 20% level due to impalatibility of the test diet. No other adverse
    findings were noted in growth, behaviour, mortality, haematology,
    urinalysis, gross pathology, organ weights, and histopathology (Kay
    and Calandra, 1962).

         Four groups of 10 male and 10 female rats received either 0 or
    10% of three different caramel colours in their diet for 90 days.
    Weight gains showed slight reduction compared with controls but food
    consumption was normal for all groups. No abnormalities were noted
    regarding haematology, urinalysis, gross and histopathology
    (Chacharonis, 1963).

         Four groups of 15 male end 15 female rats received 0, 5, 10 and
    20% of caramel colour in their diet for 90 days. No adverse effects
    were noted on appearance, behaviour, survival, body weights, food
    intake, haematology, blood chemistry, urinalysis, organ weights, gross
    and histopathology (Oser, 1963).

         Four groups of 10 male and 10 female rats received 0, 0.015, 0.3
    and 3.0% of caramel colour in their diet for 90 days. No differences
    between test and control animals were noted regarding body weight,
    food consumption, haematology, urinalysis, gross or histopathology
    (Nees, 1964).

         Four groups of rats received 0, 4, 8 and 16% caramel colour in
    their diet for three months. No convulsions or other behaviour
    abnormality or signs of neurological damage were seen. No macroscopic
    or microscopic pathological abnormalities were found in the CNS. Other
    results are still to come (Sharratt, 1971).

    Dog

         Four groups of three male and three female adult beagles received
    0, 6, 12.5 and 25% of caramel colour in their diet five days per week
    for 90 days. No significant adverse effects were noted due to the test
    compound on growth behaviour, food consumption, mortality, liver
    function, kidney function, haematology, urine analysis, gross and
    histopathology (Kay and Calandra, 1962).

    Long-term studies

    Rat

         No formal studies are available but in the course of a
    reproduction study using 25 pairs of one male and one female rat given
    0 or 0.8 g/kg body weight caramel colour in their drinking fluid as
    part of a beverage tested, several pairs survived for two years or
    longer. No deleterious effects on growth were noted. The data are
    rather incomplete (Bachmann et al., 1946).

         Six different caramels were tested in this long-term toxicity
    study. Three ammonia-sulfite process single strength (SS) and three
    ammonia-sulfite process double strength (DS) caramels each containing
    different quantities of 4-methylimidazole ranging from 200-850 ppm.
    Each group consisted of 40 male and 40 female rats (Wistar) except the
    control group, that had the double number of animals. These animals
    were selected from the first litter of parents, which had been kept on
    diets containing the various caramels since weaning age (see under
    reproduction studies). The different dose levels that were tested were
    5, 10 and 15% caramel SS and 2, 4 and 6% caramel (DS), furthermore
    three caramels SS with different methylimidazole contents, tested in
    10% and three caramels DS with different methylimidazole contents
    tested in 470 in the stock diet (see Table 1 on page 48, except that
    group 12 was not included). After one year, 10 male and female rats in
    each group were sacrificed. Findings are reported separately (see
    short-term studies).

         Observations were made of general appearance, behaviour,
    mortality, growth and food intake, haematological factors and clinical
    constituents in blood and urine. After about 14 months considerable
    mortality was observed both in control and treated groups, probably
    due to intercurrent disease. Approximately three-quarters of the
    animals died or were killed before the experiment was terminated at
    week 104. Organs of dead or killed animals were weighed and extensive
    histopathological examinations were carried out on all rats of all
    groups. However, one-third of the animals could not be examined
    histopathologically due to autolysis. At week 104 organs were weighed
    and extensive histopathological examinations were carried out on all
    rats of all groups.

         In these parameters no abnormalities were found except that the
    haemoglobin levels and haematocrit values were slightly decreased at
    week 78 and 98 in males fed the DS caramels. Leucocyte counts were
    decreased in females fed 15% SS-caramal containing 202 ppm
    4-methylimidazole and fed 10% SS-caramel containing 400 ppm
    4-methylimidazole at week 13 and 52. At autopsy an increased incidence
    of greenish discoloured mesenteric lymph nodes was observed in most
    groups fed high levels of caramel. Again microscopically an increased
    pigment-phagoeytosis in the mesenteric lymph nodes in all test groups

    (except the group with 2% caramel DS + 350 ppm 4-methylimidazole) was
    observed. No evidence of any other adverse structural or cellular
    alteration was found. Gross and microscopic examination of the other
    organs did not reveal any pathological changes attributable to the
    ingestion of the caramels. No increase in the incidence of neoplastic
    lesions in the different groups was found (Sinkeldam et al., 1976).

         Four groups of 48 male and 48 female rats (strain Wistar) were
    given diets containing 0 (control), 1, 3 and 6% of armnonia process
    caramel (ammonia catalysed "half open - half closed pan" caramel); no
    indication about the presence of 4-methylimidazole was given) for two
    years. Besides food and water intake, growth, mortality,
    haematological examination, urine analysis, kidney function tests,
    organ weights and histopathological examination were carried out.

         In both sexes, but significant in the males, there was a decrease
    in growth at all dose levels. This was accompanied by a reduction in
    the cumulative food intake. A significant reduction in white cell
    number (in the 6% group) was associated in the early part of the study
    with a lymphocytopenia, which was present until week 80 in the male
    rats fed a diet containing 3 and 6% caramel. In the female animals the
    lymphocytopenia was present until week 52 in these two groups (the 1%
    group was not tested).

         The spleen weight was reduced in a dose-related manner. The
    relative weight of the (full) caecum was clearly increased at all dose
    levels. In the histopathologieal findings a few changes are of
    interest because they may relate to the administration of the caramel.
    In the pancreas of the control animals no changes were found, while in
    the test groups (not dose-related) in total 10 hyperplastie changes
    were found. The number of tumours of the pancreas however, showed no
    relation to the administration of the caramel. There was no evidence
    of a carcinogenic effect.

         The authors concluded that a no-untoward effect could not be
    established for the caramel used in this particular study (Evans et
    al., 1976).

    OBSERVATIONS IN MAN

         In a number of animal studies a regular finding was an influence
    on the white blood cells, such as a decrease of the total number of
    leucocytes and/or a decrease in the number of lymphocytes.

         A pilot study was carried out in which daily 1.5 g of ammonia
    process caramel (prepared with ammonia by a closed pan process) was
    given to nine human volunteers for 21 days. Total circulating
    leucocytes, lymphocytes and erythrocytes together with haemoglobin
    concentration were measured prior to and during the treatment. No
    changes were found that could be attributed to caramel treatment.

         Another finding in the animal studies is an enlarged caecum and
    soft stool to diarrhoea. In this human experiment six of the subjects
    showed no differences from normal in stool frequency or condition.
    Three volunteers had occasionally soft stools (no control group was
    used) (BIBRA report 1976).

         Twenty healthy adult volunteers (10 males and 10 females) were
    given ammonia sulfite single strength caramel (no data on content of
    4-methylimidazole) for three periods of 21 days. The first period they
    received 6 g/day, the second period 12 g/day and the third period
    24 g/day. A full seven-day rest was given between each 21 day period
    of ingestion. Up till now the results of the first two periods are
    available and described here.

         Haematological, clinical biochemical and routine urinary
    parameters were monitored at the beginning of each ingestion period,
    after 10 days of ingestion and at the end of each ingestion period.
    Individual values for haemoglobin, haematocrit, RBC, corrected
    sedimentation rate, WBC and differentials (neutrophils and band stabs,
    basophils, eosinophils, monoeytes and lymphoeytes) were found to be
    within normal limits. For these values there were no changes which may
    have indicated a trend. There were a few instances of mild neutropenia
    and mild lymphocytosis but these were not consistent and were
    unrelated to ingestion of caramel. On the other hand, caramel
    ingestion was associated with increased frequency of bowel movements
    and softening or increased liquidity of faeces during the caramel
    ingestion periods, particularly in the second period when the caramel
    ingestion level doubled from 6 g/day to 12 g/day (Marier et al.,
    1977).

         Twenty healthy adult volunteers (10 males and 10 females) were
    given anmaonia sulfite double strength caramel (no data on content of
    4-methylimidazole) for three periods of 21 days. The first period they
    received 6 g/day, the second period 12 g/day and the third period
    24 g/day. A full seven day rest was given between each 21 day period
    of ingestion. Up till now the results of the first two periods are
    available and described here.

         Haematological, clinical biochemical and routine urinary
    parameters were studied at the beginning of each ingestion period,
    after 10 days of ingestion and again at the end of each ingestion
    period. Most individual values for haemoglobin, haematocrit, RBC,
    corrected sedimentation rate, WBC and differentials (Neutrophils and
    band stabs, basophils, eoslnophils, monocytes and lymphocytes) were
    found to be within normal limits. For these values there were no
    changes of clinical significance and there were no consistent patterns
    which may have suggested trends. There were a few instances of values
    outside the normal range indicating mild neutropenia and mild
    lymphocytosis but these were observed throughout the two periods
    including pre-ingestion time, only at pre-ingestion or only as
    isolated incidents. These changes are not believed to be related to

    the ingestion of ammonia sulfite caramels. Many subjects experienced
    frequency of evacuation and softening or increased liquidity of faecal
    discharges during the two caramel ingestion periods and particularly
    in the second period when the caramel ingestion level was doubled from
    6 g/day to 12 g/day (Marier et al., 1977a).

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    Unpublished BIBRA report (1976) A study of the haematological effects
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    Brusick, D. (1974) Mutagenic evaluation of compound FDA 71-83,
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    See Also:
       Toxicological Abbreviations