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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