CARAMEL COLOURS
EXPLANATION
In the period since 1972, when the fifteenth report of the
Committee was published (Annex 1, reference 26), caramel colours have
been classified into 4 classes which differ in their method of
manufacture, composition, functional properties, and application.
1. Caramel Colour I (synonyms: plain caramel, caustic caramel, and
spirit caramel); this class is prepared by the controlled heat
treatment of carbohydrates with alkali or acid.
2. Caramel Colour II (synonyms: caustic sulfite process caramel); this
class is prepared by the controlled heat treatment of carbohydrates
with sulfite-containing compounds.
3. Caramel Colour III (synonyms: ammonia caramel, ammonia process
caramel, closed-pan ammonia process caramel, open-pan ammonia process
caramel, bakers' caramel, confectioners' caramel, and beer caramel);
this class is prepared by the controlled heat treatment of
carbohydrates with ammonium compounds.
4. Caramel Colour IV (synonyms: ammonia sulfite process caramel,
sulfite ammonia caramel, sulfite ammonia process caramel, acid-proof
caramel, beverage caramel, and soft-drink caramel); this class is
prepared by the controlled heat treatment of carbohydrates with
ammonium-containing and sulfite-containing compounds.
Caramel colours were reviewed at the eighth, thirteenth,
fifteenth, sixteenth, eighteenth, twenty-first, and twenty-fourth
meetings of the Committee (Annex 1, references 8, 19, 26, 30, 35, 44,
& 53). The thirteenth meeting concluded that a toxicological
distinction between caramels produced commercially and caramel formed
in cooked foods or when sucrose is heated is unwarranted except with
caramel prepared by processes using ammonia or ammonium salts. This
conclusion was endorsed by the fifteenth meeting and an ADI "not
limited" was allocated to caramel colours prepared by processes other
than those involving ammonia or ammonium salts; a temporary ADI of
0-100 mg/kg b.w. was allocated to caramel colours produced by the
ammonia process. The sixteenth meeting reviewed further information on
the composition of caramel colours produced by the ammonia process,
including the 4-methylimidazole content. Revised specifications were
prepared and the previously-established temporary ADI was maintained.
The specifications for this class were further revised by the
eighteenth meeting and the temporary ADI was extended pending the
results of long-term and reproduction studies on caramel colours
prepared by the ammonia or ammonia sulfite process.
The twenty-first meeting of the Committee noted that the
specifications for caramel colour (ammonia process) were ambiguous,
since they appeared to cover caramel colours manufactured by the
ammonia sulfite process as well. Separate specifications were prepared
for caramel colour (ammonia process) and caramel colour (ammonia
sulfite process), which have since been designated caramel colours III
and IV, respectively. The two classes have been shown to differ in
toxicity. The principal toxic effect of caramel colour III is
depression of circulating lymphocytes and leucocytes, and a no-effect
level could not be determined; accordingly, the temporary ADI was
revoked. The temporary ADI of 0-100 mg/kg b.w. was retained for
caramel colour IV pending the submission of reports of adequate
carcinogenicity/teratogenicity studies.
The twenty-fourth meeting extended the temporary ADI for caramel
colour IV and confirmed that caramel colour II had no ADI since it was
not included in the ADI for caramel colour I nor the temporary ADI for
caramel colour IV.
Since the previous evaluations, additional data have become
available and are summarized and discussed in the following
monographs. Previously-published monographs have been expanded and are
reproduced in their entirety under the class of caramel colour to
which they relate.
CARAMEL COLOUR I
EXPLANATION
At the thirteenth and fifteenth meetings (Annex 1, references 19
& 26), the Committee concluded that caramel colour I is a natural
constituent of the diet and is acceptable as an additive. An ADI "not
limited" was allocated at the fifteenth meeting.
BIOLOGICAL DATA
Biochemical aspects
No information available.
Toxicological studies
Special study on mutagenicity
Two samples of caramel colour I with different colour intensities
were subjected to the Ames test using Salmonella typhimurium strains
TA98, TA100, TA1535, TA1537, and TA1538. Caramel colour I was neither
mutagenic nor cytotoxic, either with or without activation by rat
liver S-9 fraction, at concentrations up to 20 µl per plate (Richold &
Jones, 1980a,b).
Acute toxicity
No information available.
Short-term studies
Rats
Caramel colour I was administered to groups of 20 weanling female
Wistar rats at dietary levels of 0, 15, or 30% for 8 weeks followed by
a 4-week recovery period. Diarrhoea was observed in the treated
animals and food efficiency was decreased, but the growth rate was
normal. Haematological indices, in particular leucocyte counts, were
normal throughout the study. The relative caecal weights were
increased after eight weeks, but returned to normal by the end of the
4-week recovery period. Discolouration of the mesenteric lymph nodes
was observed in animals of both treatment groups after eight weeks,
but the discolouration diminished during the recovery period. No other
gross or microscopic pathological changes were reported (Sinkeldam &
van der Heyden, 1976).
Long-term studies
No information available.
Observations in man
No information available.
Comments
Caramel colour I is free of the heterocyclic compounds associated
with convulsant activity or depressed lymphocyte counts which occur in
caramels prepared using ammonia or ammonium salts, and displays a low
order of short-term toxicity. None of the data suggest a need to
revise earlier JECFA recommendations.
EVALUATION
Estimate of acceptable daily intake for man
ADI "not specified".
REFERENCES
Richold, M. & Jones, E. (1980a). Ames metabolic activation test to
assess the potential mutagenic effect of ETA-38-2H. Unpublished
report No. FDC 8/80359 from Huntingdon Research Centre,
Huntingdon, England. Submitted to WHO by International Technical
Caramel Association.
Richold, M. & Jones, E. (1980b). Ames metabolic activation test to
assess the potential mutagenic effect of ETA-38-1W. Unpublished
report No. FDC 8/80361 from Huntingdon Research Centre,
Huntingdon, England. Submitted to WHO by International Technical
Caramel Association.
Sinkeldam, E.J. & van der Heyden, C.A. (1976). Short-term feeding
test with three types of caramels in albino rats. Unpublished
report No. R4789 from CIVO/TNO, Zeist, The Netherlands. Submitted
to WHO by International Technical Caramel Association.
CARAMEL COLOUR II
EXPLANATION
The twenty-fourth meeting of the Committee (Annex 1, reference
53) drew attention to the fact that caramel colour II has no ADI since
it is not included in the ADI of caramel colour I. This material has
not previously been evaluated by the Committee.
BIOLOGICAL DATA
Biochemical aspects
No information available.
Toxicological studies
Special studies on mutagenicity
Caramel colour II at concentrations of 2.5-20 µl/plate was
neither mutagenic nor cytotoxic in the Ames test using Salmonella
typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538, with or
without metabolic activation by rat liver S-9 fraction (Richold &
Jones, 1980).
In a similar test, caramel colour II was neither mutagenic nor
cytoxic at concentrations of 50-5000 µg/plate (Richold et al.,
1984).
Caramel colour II was tested for potential mutagenic activity
based on induction of DNA repair (unscheduled DNA synthesis) in
cultured human epithelial (HeLa 53) cells. Caramel colour II
was incorporated in the culture medium at concentrations of
25-51,200 µg/ml and the test was performed on 2 occasions both in the
presence and absence of rat liver S-9 mix. In both tests, in the
absence of the S-9 mix a small but statistically significant increase
in the number of silver grains over nuclei was observed at a
concentration of 25,600 µg/ml; no significant increases were seen at
higher concentrations in this test nor at any concentration in the
repeat test (Allen & Proudlock, 1984).
Caramel colour II was not clastogenic to cultured Chinese hamster
ovarian cells at concentrations of 500, 2500, or 5000 µg/ml either in
the presence or absence of rat liver S-9 mix (Allen et al., 1984).
Acute Toxicity
No information available.
Short-term studies
Rats
Five groups of 20 male and 20 female weanling Fischer F-344 rats
were given caramel colour II in drinking water at dose levels of 0, 4,
8, 12, or 16 g/kg b.w./day for 90 days. Body weights and food and
water intakes were recorded weekly. Haematological examinations, blood
bichemical studies, and urinalysis were performed during the seventh
week and at termination on all animals. Animals were fasted overnight
prior to bleeding from the orbital sinus under anaesthesia; the
animals were also fasted during the 24-hour urine collection period.
At necropsy, organs were weighed and all animals were examined
macroscopically. Histopathological examinations were conducted on all
animals in the control and high-dose groups and on animals from the
low- and mid-dose groups that died or were sacrificed in extremis.
All animals showed a weight loss and reduced food intake between
weeks 7 and 8, presumably due to the fasting prior to bleeding and
during urine collection. In addition, caramel colour II caused a dose-
related reduction in body-weight gain and in food and fluid intake in
both sexes. Males treated with 8 g caramel colour II per kg b.w., and
females treated with 4 and 8 g/kg b.w., had significantly lower food
consumption than controls for 4 of 13 weeks, 3 of 13 weeks, and 6 of
13 weeks, respectively; mean food consumption, of both males and
females treated with 12 and 16 g/kg b.w., was significantly lower than
food consumption of their respective controls. All treated groups had
lower mean fluid intake than their respective controls, usually
significantly lower. Males receiving 12 and 16 g/kg b.w. had
significantly lower mean body weights than controls from weeks 7-13
and weeks 4-13, respectively. Females treated with 8 g/kg b.w. had
significantly lower mean body weights than controls from week 11-13
while, beginning at week 7, females receiving 12 and 16 g/kg b.w. had
lower mean body weights than controls.
Occasional statistically-significant changes were seen in some
haematological and clinical chemical parameters, but the mean values
were within the normal range for Fischer 344 rats and were not
considered to be of toxicological significance. There was a dose-
related reduction in urinary volume and pH, and an increase in urine
specific gravity.
At necropsy, treatment-related increases were observed in kidney
weights and in full and empty caecum weights, but no significant
histopathological changes were observed in these or other tissues.
Dose-related staining of the gastrointestinal tract and mesenteric
lymph nodes was noted, and deposits of yellow pigment were seen
histopathologically in the caecal submucosa and mesenteric lymph nodes
of the top-dose groups (the only treated animals examined
comprehensively). Reactive hyperplasia was not associated with the
pigment deposition.
The investigators concluded that the reduced body weights, food
and fluid intakes, and increased kidney weights were due to water
imbalance reflecting poor palatability of the drinking solution rather
than toxic effects of caramel colour II per se (MacKenzie, 1985).
Comments
The above data are insufficient to evaluate caramel colour II for
an ADI, but the substance has a low sub-chronic toxicity and no
frankly pathological effects were seen in the 90-day study.
EVALUATION
Estimate of acceptable daily intake for man
No ADI allocated.
REFERENCES
Allen, J.A., Brooker, P.C., Birt, D.M., & McCaffrey, K.J. (1984).
Analysis of metaphase chromosomes obtained from CHO cells
cultured in vitro and treated with caramel colour (II).
Unpublished report No. ITC 3A/84965 from Huntingdon Research
Centre, Huntingdon, England. Submitted to WHO by International
Technical Caramel Association.
Allen, J.A., & Proudlock, R.J. (1984). Autoradiographic assessment of
DNA repair in mammalian cells after exposure to caramel colour
II. Unpublished report No. ITC 2A/84750/2 from Huntingdon
Research Centre, Huntingdon, England. Submitted to WHO by
International Technical Caramel Association.
MacKenzie, K.M. (1985). 90-day toxicity study of caramel color
(caustic sulfite process) in rats. Vols. I & II. Unpublished
report No. 6154-105 from Hazleton Laboratories America Inc.,
Madison, WI, USA. Submitted to WHO by International Technical
Caramel Association.
Richold, M. & Jones, E. (1980). Ames metabolic activation test to
assess the potential mutagenic effect of ETA-38-3N. Unpublished
report No. FDC 8/80356 from Huntingdon Research Centre,
Huntingdon, England. Submitted to WHO by International Technical
Caramel Association.
Richold, M., Jones, E., & Fenner, L.A. (1984). Ames metabolic
activation test to assess the potential mutagenic effect of
caramel colour II. Unpublished report No. ITC 1A/84708 from
Huntingdon Research Centre, Huntingdon, England. Submitted to WHO
by International Technical Caramel Association.
CARAMEL COLOUR III
EXPLANATION
In earlier evaluations, the presence of 4-methylimidazole in
caramel colour III was noted, particularly since this compound was
likely to be the causal agent of convulsions in cattle and sheep fed
ammonia-treated molasses. However, at its twenty-first meeting, the
Committee (Annex 1, reference 44) no longer considered this a cause
for concern since the introduction of chemical specifications limits
the concentration of 4-methylimidazole in caramel colour III. The
twenty-first meeting identified the principal toxic effect of
ammoniated caramels as the depression of circulating lymphocytes and
total leucocytes for which a no-effect level had not been determined;
consequently, the temporary ADI for caramel colour III was revoked.
BIOLOGICAL DATA
Biochemical aspects
Absorption, distribution, and excretion
In groups of 2-4 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 & Wynn, 1951).
Toxicological studies
Special study on 4-methylimidazole (4-MeI)
4-MEI has been shown to be the most likely toxic component in
ammoniated molasses, its being a convulsant to rabbits, mice, and
chicks at oral doses of 360 mg/kg b.w. (Nishie et al., 1969).
Mice
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 the following table:
Convulsant and lethal effects of imidazoles
CD50±SE LD50±SE
in mg/kg in mg/kg b.w.
b.w i.p oral b.w. i.p 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 1,800 ± 45 610 ± 7.4 1,880 ± 45
All the imidazoles tested produced varying degrees of tremor,
running, restlessness, dialorrhea, Straub tail, opisthotonus and tonic
extensor seizure that ended in death (Nishie et al., 1970).
Chickens
The CD50 and the LD50 of 4-MEI by i.p. injection in 1-day-old
chicks were 174±10 mg/kg b.w. and 210±15 mg/kg b.w., respectively.
Orally, the CD50 was 580±30 mg/kg b.w. and the LD50 was 599±50 mg/kg
b.w. Doses of 100 mg/kg b.w. i.p. caused tremors, peeping, and
spreading of the wings. Doses over 150 mg/kg b.w. i.p. caused
opisthotonus, prostration with clonic leg movements, and terminal
tonic extensor seizure (Nishie et al., 1970).
Special studies on reduction of total lymphocyte counts
Following the request in the twenty-first report of the Committee
for information on the factor(s) responsible for the haematological
effects of caramel colour III, a series of studies have been performed
to investigate the mechanism(s) underlying these effects and the
agent(s) responsible.
Rats
The effects of vitamin E, folic acid, pyridoxine and choline on
the capacity of caramel colour III to reduce total lymphocytes in the
blood of rats fed caramel colour III were examined. Four groups of 10
male weanling rats were fed Spratt's diet containing 8% caramel colour
III and supplemented with vitamin E (100 mg/kg), folic acid
(10 mg/kg), pyridoxine hydrochloride (10 mg/kg), or choline chloride
(1000 mg/kg). Groups on Spratt's diet alone or on Spratt's diet with
8% caramel colour III without any supplement were used as controls.
After 12 days, there was a marked reduction in total white blood cells
and lymphocytes in rats fed the diet containing 8% caramel colour III
and neutrophil counts were increased. Dietary supplementation with
vitamin E, folic acid, or choline did not noticeably affect lymphocyte
counts. However, rats receiving the diet supplemented with pyridoxine
had white blood cells and lymphocytes in numbers similar to those fed
the basal diet without caramel colour III. The basal diet was found to
contain 2.3 mg/kg pyridoxine.
In a further study using CIVO stock diet (pyridoxine
concentration 3 mg/kg), rats receiving 8% caramel colour III in the
diet showed a reduction in total white blood cell and lymphocyte
numbers; these changes were ameliorated by the addition of 10 mg/kg
pyridoxine in the diet. In this study, neutrophils were not affected
by the caramel colour III treatment, but the plasma pyridoxal
phosphate levels were reduced by treatment (Sinkeldam et al., 1984).
In order to quantify the relationship between dietary pyridoxine
and reduction of lymphocyte counts by caramel colour III, and to study
the effects of age, two 14-day studies were performed, one in
weanling, and the second in mature, Wistar rats; in other respects the
protocols were identical. Groups of 10 male animals were fed diets
containing approximately 2.5, 6, 12, or 24 ppm of pyridoxine. At each
dietary level groups were given caramel colour III1 in the drinking
water at levels of 0, 1, 4, or 8%. A clear inverse dose relationship
between the severity of lymphocyte depression and the pyridoxine level
of the diet was observed. In weanling rats fed a diet containing
2.5 ppm pyridoxine, statistically significant lymphocyte reduction
occurred at all caramel colour III levels on days 6 and 13. Groups fed
diets containing 6, 12, or 24 ppm pyridoxine did not show
statistically significant reductions in lymphocyte counts on day 6.
However, on day 13, groups receiving 4 or 8% caramel colour III and 6
ppm pyridoxine in the diet had a statistically significant reduction
in lymphocytes. On day 13 at a dietary level of 12 ppm pyridoxine only
the 8% caramel colour III group had a statistically significant
reduction in lymphocytes. There were no significant reductions in
lymphocyte counts in animals fed 24 ppm pyridoxine at any level of
caramel colour III intake. In mature rats fed 4 and 8% caramel colour
III a statistically significant reduction in lymphocytes occurred at
dietary pyridoxine levels of 12 ppm and lower on day 6. On day 13
lymphocytes were significantly decreased at caramel colour III doses
1, 4, and 8% in animals fed a diet containing 2.5 ppm pyridoxine.
There were no significant decreases in lymphocytes at these dose
levels of caramel colour III in animals fed 6, 12, or 24 ppm
pyridoxine (Sinkeldam, 1981; 1982a).
1 This sample of caramel colour III contained 107 mg/kg
2-acetyl-4(5)-tetrahydroxybutylimidazole (THI) on a colour
equivalent basis, 204 mg THI/kg on an 'as is' basis, and 295 mg
THI/kg on a solids basis.
Special studies on 2-acetyl-4(5)-tetrahydroxybutylimidazole (THI)
An isolation procedure was developed and a fraction of caramel
colour III that contained the lymphocyte-depressing activity was
isolated. The single component of this fraction that was responsible
for the activity was identified as THI (Kroplien et al., 1984).
Rats
THI was administered to groups of 10 male weanling Wistar rats at
levels of 0, 2, 5, or 20 ppm in drinking water for 7 days; a fifth
group received a 1% solution of caramel colour III in drinking water
as a positive control. THI produced a marked depression of lymphocyte
counts at all dose levels and a dose-dependent increase in
neutrophils. The lymphocyte-depressing potency of 2 ppm THI was
comparable to that of 1% caramel colour III in the drinking water,
indicating a level of approximately 200 mg/kg THI in the caramel
sample. This compares with the result of chemical analysis of a sample
of this batch of caramel colour III, which indicated a value of
204 mg/kg THI on an 'as is' basis (Sinkeldam, 1982b; Kroplien, 1984).
Special studies on carcinogenicity (see also long-term studies)
Mice
Three groups of 50 male and 50 female B6C3F1 mice were given
drinking water containing 0, 1.25, or 5% caramel colour III for 96
weeks followed by 8 weeks of drinking water without caramel colour;
the animals were fed CFR diet ad libitum. The caramel colour III
used in this study contained less than 25 mg/kg THI.
The animals were observed daily for abnormalities and mice
showing signs of ill-health were isolated to be returned to the group
if their condition improved but otherwise killed and autopsied.
Individual body weights were recorded weekly for the first 14 weeks,
then every-other-week. Food and water consumption were recorded over a
2-day period before each weighing. During week 104, fresh urine
samples were obtained from all survivors and analysed for pH, protein,
glucose, bilirubin, ketones, occult blood, and urobilinogen. At
termination the animals were killed by exsanguination under ether
anaesthesia and haematological examinations were performed, which
consisted of measurements of haemoglobin concentrations, haematocrit
values, erythrocyte counts, leucocyte counts, platelet counts, and
differential leucocyte counts. At autopsy, gross findings were
recorded and the following organs weighed: brain, heart, liver,
spleen, adrenals, and testes or ovaries. Samples of these organs and
of the salivary gland, trachea, lungs, thymus, lymph nodes, stomach,
small intestine, pancreas, urinary bladder, pituitary, thyroid,
prostate, seminal vesicle, uterus, mammary gland, skeletal muscle,
eye, Harderian glands, spinal cord, sciatic nerve, and any other
tissues of abnormal appearance were examined histologically
(haematoxylin- and eosin-stained). Histopathological examinations were
also performed on mice that died and on those that were killed in
moribund condition. During the in-life phase no consistent differences
were noted between the test and control groups with respect to growth
or water intake.
The cumulative mortality of males given 5% caramel colour III was
higher than that of controls from week 100 to the end of the
experiment, but there were no clear pathological differences in any
organs and no treatment-related abnormalities in urinalyses.
The only statistically significant differences in haematological
parameters between control and treated groups were elevations of the
total leucocyte counts in males of both treatment groups, but the
observed values were within the range encountered for the strain of
B6C3F1 mice used in the study. No treatment-related gross pathology
was noted during or at the end of the experiment. Malignant lymphoma/
leukaemia, hepatocellular carcinoma, and sub-cutaneous fibrosarcoma
and/or malignant fibrous histocytoma were frequent, but no significant
differences were found in their incidences between treated and control
groups. Adenomas and adenocarcinomas of the lungs, hyperplastic
nodules of the liver, and fibroma of the sub-cutis were frequent in
males, but their incidences were similar in treated and untreated
mice.
The authors concluded that, under the conditions used, caramel
colour III was not carcinogenic for B6C3F1 mice (Hagiwara et al.,
1983).
Rats
Three groups of 50 male and 50 female F344 rats were given
caramel colour III in drinking water at levels of 0, 1, or 4% for 104
weeks followed by drinking water without caramel for 9 weeks. The
animals were fed ad libitum basal diet that contained 11-12 mg/kg
pyridoxine, and the caramel colour III used contained less than
25 mg/kg THI. During the experimental period, all animals were
observed daily, and clinical signs and mortality were recorded. Body
weights were recorded weekly during the first 13 weeks of the study
and then every 4 weeks. Moribund or dead animals and animals
sacrificed at termination were autopsied and examined for the
development of tumours in the following organs and tissues: brain,
pituitary, thyroid (including parathyroid), thymus, lungs, trachea,
heart, salivary glands, liver, spleen, kidneys, adrenals, tongue,
oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum,
urinary bladder, lymph nodes, pancreas, gonads, accessory genital
organs, mammary gland, skin, musculature, peripheral nerve, spinal
cord, sternum, femur, eyes, ear duct, and nasal cavity. These tissues
were also examined histologically (haematoxylin- and eosin-stained).
No treatment-related differences in growth or survival rates were
noted. No dose-related effects were found either in the incidence or
induction time of tumours in the various organs and tissues except in
the pituitary gland of males of the top-dose group in which the
incidence of tumours was significantly higher than that in controls.
However, pituitary tumours are among the most common spontaneous
tumours in F344 rats that occur with variable incidence; most of the
tumours were microscopic and there were no significant differences in
their induction times compared with controls. The authors concluded
that the higher incidence of pituitary tumours was not related to
caramel administration, but could be explained by the variability of
spontaneous tumour incidence (Maekawa et al., 1983).
Special studies on mutagenicity
Caramel colour III (a blend of 3 commercial samples) was
evaluated by the Ames Salmonella/microsome plate test and the
Saccharomyces/microsome plate test. The Salmonella test organisms
used were TA98, TA100, TA1535, TA1537, and TA1538; the Saccharomyces
was strain D4, and the tests were conducted in the presence and
absence of liver S-9 mix from Araclor-induced rats. The dose range
employed was 1-50 mg/plate for TA100 and 1-20 mg/plate for all other
test strains. Caramel colour III was non-mutagenic to the test
organisms under these conditions (Jagannath & Brusick, 1978a).
In a similar study using the same Salmonella tester strains,
caramel colour III was non-mutagenic in a range of concentrations up
to 20 µl/plate with or without metabolic activation (Richold & Jones,
1980).
Caramel colour III was non-mutagenic in the Ames test against
Salmonella strains TA98 and KTA100 at concentrations of
0-1 mg/plate, with or without metabolic activation. Acidic and basic
organic extracts of the caramel were also non-mutagenic (Ashoor &
Monte, 1983).
Thirteen commercial caramel colours (not identified) were
examined for mutagenicity in the Ames test, using Salmonella strains
TA98 and TA100 with and without metabolic activation, and for
DNA-damaging effects in E. coli (Wild/pol A-, Wild/rec A-); none
of the samples tested was active under the test conditions (Kawana
et al., 1980).
Five samples of commercial caramel colour (not identified) were
tested for mutagenicity in the Ames test using Salmonella strains
TA98 and TA100. All samples gave equivocal results with TA100 and 2 of
the 5 samples were equivocal with TA98 (Kawachi et al., 1980).
Five samples of caramel colour were tested in the Ames
Salmonella plate assay using strains TA98, TA100, and TA1537 without
metabolic activation but with a 20-minute preincubation step. The same
samples were used in chromosome aberration tests performed on a
cultured Chinese hamster lung fibroblast cell line, both in the
presence and absence of S-9 fraction. All samples of caramel colour
were designated as positive both in the Ames assay and in the
chromosome aberration test (aberrations in 20% of metaphase cells)
(Ishidate & Yoshikawa, 1980).
The same series of caramel colour samples was tested by Jagannath
and Brusick, and all were found to be non-mutagenic in the Ames test
using Salmonella strains TA98, TA100, TA1535, TA1537, and TA1538,
and Saccharomyces strain D4, in the presence or absence of S-9 mix.
One of the samples in this study and in the Ishidate & Yoshikawa
(1980) study was caramel colour III (Jagannath & Brusick, 1978b).
The mutagenicity of a series of samples taken at various stages
in the manufacture of caramel colour III were assayed in the Ames test
using Salmonella strains TA98, TA100, and TA1535, with and without
metabolic activation. No mutagenicity was seen with any sample against
all three tester strains in the presence of S-9 fraction but and
increased number of revertants was found with strain TA100 in the
absence of S-9 fraction. Mutagenic activity was associated with
samples taken late in the process (Jensen et al., 1983).
A sample of caramel colour III was non-mutagenic in the Ames test
against Salmonella strains TA98, TA100, TA1535, TA1537, and TA1538
both in the presence and absence of rat hepatic S-9 fraction at
caramel colour concentrations up to 5 mg/plate (Richold et al.,
1984).
The same sample as that used by Richold et al. (1984b) was
tested for potential mutagenic activity based on induction of
unscheduled DNA synthesis in cultured human epithelial (HeLa 53) cells
both in the presence and the absence of rat liver S-9 fraction. The
test was performed on two occasions at caramel colour III
concentrations of 25-51,200 µg/ml in the culture medium. In both
tests, caramel colour III caused a significant increase in the number
of silver grains found over cell nuclei at concentrations of 6,400 and
12,800 µg/ml in the absence of S-9 mix; significant increases were not
observed at higher or lower concentrations in the absence of S-9 mix
nor at any of the concentrations tested in the presence of S-9 mix.
The authors concluded that, although reproducible effects on
unscheduled DNA synthesis were demonstrated in the test, this effect
may be moderated by metabolism (Allen & Proudlock, 1984).
Caramel colour III was not clastogenic to cultured Chinese
hamster ovarian cells at concentrations of 500, 2500, or 5000 µg/ml
either in the presence or absence of rat liver S-9 mix (Allen
et al., 1984).
The clastogenicity of caramel colour III was evaluated in an
in vitro cytogenetic assay using cultured Chinese hamster ovarian
cells. A significant increase in chromosome aberrations was observed
at concentrations of 3 mg/ml or higher in the absence of metabolic
activation; similar findings were reported for sodium ascorbate at
2 mg/ml (Galloway & Brusick, 1981a).
These tests were repeated in the presence of rat liver S-9
fraction, and neither caramel colour III at a concentration up to
5 mg/ml nor sodium ascorbate at a concentration of 5 mg/ml was active
(Galloway & Brusick, 1981b).
In an in vivo mouse micronucleus test, caramel colour III was
administered by gavage to 5 males and 5 females at doses of 0, 1.05,
or 3.5 g/kg b.w. (2 doses 24 hours apart). Caramel colour III did not
increase the incidence of micronuclei in polychromatic erythrocytes
obtained from marrow and was considered by the authors not to exhibit
clastogenic activity under the conditions of the test (Cimino &
Brusick, 1981).
Special studies on teratogenicity
Teratogenicity studies were carried out on caramel colour III in
mice, rats, and rabbits. The doses employed were 0, 16, 74.3, 345, and
1600 mg/kg b.w. in all three species.
Mice
Caramel colour III was administered by gavage to groups of 22 or
23 pregnant CD1 mice at the above doses beginning on day 6 and
continuing through day 15 of gestation. On day 17, all dams were
subjected to Caesarian section under anaesthesia and the numbers of
implantation sites, resorption sites, and live and dead foetuses were
recorded. The urogenital tract of each dam was examined for anatomical
abnormalities and all foetuses were examined grossly for external
abnormalities. One-third of the foetuses from each litter were
examined for visceral abnormalities using the Wilson technique, and
the remaining two-thirds were examined for skeletal defects after
clearing and staining with Alizarin Red. Caramel colour III had no
clearly discernible effects on nidation nor on maternal or foetal
survival. The number of abnormalities of either soft or skeletal
tissues of the test groups did not differ from those occurring
spontaneously in controls (Morgareidge, 1974).
Rats
Caramel colour III was administered by gavage to groups of 21-24
pregnant Wistar rats at the above doses beginning on day 6 and
continuing daily through day 15 of gestation. On day 20, all dams were
subjected to Caesarian section under anaesthesia and the numbers of
implantation sites, resorption sites, and live and dead foetuses were
recorded. The body weights of live pups were also recorded. All
foetuses were examined grossly for external abnormalities. One-third
of the foetuses from each litter were examined for visceral
abnormalities using the Wilson technique, and the remaining two-thirds
were examined for skeletal defects after clearing and staining with
Alizarin Red. Caramel colour III had no clearly discernible effect on
nidation nor on maternal or foetal survival. The number of
abnormalities seen in either soft or skeletal tissues of the test
groups did not differ from those occurring spontaneously in controls
(Morgareidge, 1974).
Rabbits
Caramel colour III was administered by gavage to groups of 11 or
12 pregnant Dutch-belted female rabbits at the above doses beginning
on day 6 and continuing daily through day 18 of pregnancy. On day 29,
all does were subjected to Caesarian section under anaesthesia and the
numbers of corpora lutea, implantation sites, resorption sites, and
live and dead foetuses were recorded. All foetuses were examined
grossly for the presence of external congenital abnormalities. The
live foetuses from each litter were then placed in an incubator for 24
hours for evaluation of neonatal survival. Surviving pups were
sacrificed and all pups examined for visceral abnormalities by
dissection. All foetuses were then cleared and stained with Alizarin
Red and examined for skeletal defects. Caramel colour III treatment
had no effect on nidation nor on maternal or foetal survival. The
number of abnormalities in either soft or skeletal tissues of the test
groups did not differ from those occurring spontaneously in controls
(Morgareidge, 1974).
Acute toxicity
LD50
Species Route (mg/kg b.w.) Reference
Rat oral > 2.3 ml approx. or eq. 1,900 Foote et al., 1958
Rat oral > 25 ml approx. or eq. 17,500 Chacharonis, 1960
Rat oral > 30 ml approx. or eq. 20,400 Chacharonis, 1963
No treatment-related effects were detected during the observation
of animals for 14 days after administration of single doses of 12
different caramels manufactured with ammonia or sulphate ammonia
catalysts (Foote et al., 1958; Chacharonis, 1960; 1963). Single
doses of caramel colour III of up to 10 g/kg b.w. in mice and 15 g/kg
b.w. in rabbits did not cause convulsions or others signs of distress
(Sharratt, 1971).
Short-term studies
Mice
In a 4- to 6-week study, albino Swiss mice (10 males and 10
females/group) were fed caramel colour III containing 830 ppm
4-methylimidazole at concentrations of 0, 1, 2, 4, 8, or 16% in the
diet. No influence of caramel colour III on appearance, behaviour, or
food intake was observed. Growth was decreased, especially in the
third and fourth weeks, in the groups fed 16% caramel colour III. The
faeces of the animals fed the higher dose levels were soft, tarry in
appearance, poorly-formed, and sticky or pasty in consistency.
In the males fed 16% caramel colour III, an increase of
neutrophilic leucocytes and a decrease in lymphocytes were observed.
The mean relative weights of the caeca (full and empty) were increased
at the 4, 8, and 16% dietary levels. No other remarkable findings were
observed on gross examination; histopathological examinations were not
conducted. No information is available on the THI content of the
sample of caramel colour III used in this study, nor on the dietary
levels of pyridoxine (Procter, 1976).
In a pilot study to select the dose levels for a carcinogenicity
study, B6C3F1 mice were fed caramel colour III for 13 weeks. No
details of this study were reported (Hagiwara et al., 1983).
Rats
Four groups of 10 male and 10 female rats received either 0 or
10% of 2 different samples of caramel colour III in their diet for 90
days. Weight gains showed slight reductions compared with controls,
but food consumption was normal in all groups. No abnormalities were
noted regarding haematology, urinalysis, gross pathology, or
histopathology. No information is available on the THI content of the
samples of caramel colour III used in this study (Charcharonis, 1963).
Four groups of rats received 0, 4, 8, or 16% caramel colour III
in their diet for 3 months. No convulsions or other behavioural
abnormality or signs of neurological damage were seen. No macroscopic
pathological abnormalities were found in the central nervous system
(Sharratt, 1971).
Six groups of 15 rats (CFE strain) of each sex were fed diets
containing 4, 8, or 16% caramel colour III produced by either an
"open" or a "closed" pan process for 13 weeks; a group of 25 rats of
each sex served as controls. Body-weight gain was decreased at all
dietary levels of both caramel colours. Haemoglobin concentrations
were reduced at the highest dietary levels at week 6, while at the
lower levels this effect was less clear. After 13 weeks, the males
at all dose levels had significantly decreased haemoglobin
concentrations, but this effect occurred in females only at the 8 and
16% levels. In some groups there was a less consistent decrease in the
total number of red blood cells at 6 and 13 weeks. The total number of
leucocytes was significantly decreased and a lymphocytopenia was
present at all dose levels at 6 and 10 weeks; at 13 weeks these
changes were observed only in males. At necropsy, decreased weights of
the thymus and spleen were observed at the 8 and 16% dose levels. The
caecal weights were increased at the 8 and 16% dose levels compared
with 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 6-hour period without
water or in the 2-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 of the THI
content of the caramel colours used in this study were available
(Gaunt et al., 1975).
Seven groups of 20 female Wistar rats were fed diets containing
0, 15, or 30% of 3 types of caramel colour, one of which was a sample
of caramel colour III, for 8 weeks followed by a 4-week recovery
period. The diets containing caramel colour III caused dose-related
decreases in body weights and food efficiency, and also caused
diarrhoea at the 30% dietary level. Leucocyte counts at 4 weeks were
significantly increased at the highest dose level whereas at 8, 10,
and 12 weeks no differences were observed between test groups and
controls. The relative weights of the caeca of animals receiving
caramel colour III, both filled and empty, were increased at 4 and 8
weeks but reverted to normal after the 4-week recovery period.
Gross examination at autopsy after 8 weeks revealed a slight,
dose-related brown discolouration of the mesenteric lymph nodes in a
few animals of each test group. After recovery periods of 2 or
4 weeks the discolouration 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 2 or 4 weeks. No details of the
THI content of the caramel colour III used in this study were
available (Sinkeldam & van der Heyden, 1976a).
In a 10-week feeding study, groups of 15 male and 15 female
Sprague-Dawley rats were fed diets containing caramel colour III at
concentrations of 0, 1.25, 2.5, 5.0, 10.0, or 15.0%. In the animals
receiving caramel colour III at levels of 5% or higher, the faeces
became soft within two weeks and the water content of the faeces was
higher than the faeces of controls. Body-weight gains were generally
reduced in animals fed caramel colour III, particularly during the
last 2-4 weeks of the study.
In rats fed caramel colour III, there were no changes in
haemoglobin levels or erythrocyte counts; a significant reduction in
lymphocyte counts and a coincident increase in the number of segmented
neutrophils was observed at all dose levels. No macroscopic evidence
of abnormal pigmentation of the mesenteric lymph nodes was found. An
increase in empty caecal weight was consistently evident in animals of
both sexes given caramel colour III at the 5, 10, and 15% levels.
Histopathological examination did not reveal changes in the
structure of the ileal or caecal mucosa nor in the reticuloendothelial
components of the central or peripheral systems. No abnormal
pigmentation of the lymph nodes was found. No details of the THI
content of the caramel colour used in this study were available
(Procter et al., 1976).
Groups of 10 male and 10 female Wistar rats were fed diets
containing caramel colour III at concentrations of 0, 1.25, 2.5, 5.0,
10.0, or 15.0% for 10 weeks. Caramel colour III caused loose stools,
particularly at the 5% dietary level and body weights were slightly
decreased in both sexes at this level. Leucocyte (lymphocyte) counts
were decreased in males and in females fed 15% caramel colour III; at
lower dose levels this effect occurred only in females. The relative
weight of the caecum (both full and empty) was increased by feeding
caramel colour III. Minimal amounts of pigment were observed in
mesenteric lymph nodes of several rats fed 1.25% and higher levels of
caramel colour III. From this experiment it appeared that caramel
colour III was more active than two other types of caramel tested
concurrently in regard to growth depression, enlargement of the
caecum, and decrease in leucocyte counts. No details of the THI
content were available (Sinkeldam & van der Heyden, 1976b).
In a 10-week study, weanling Wistar rats were fed caramel colour
III in the diet at levels of 0, 0.5, 1.0, 2.0, 4.0, or 16%. There were
15 males and 15 females in each group except for the control group,
which had 60 animals of each sex, and the highest dose group, which
had 10 animals of each sex. Food intake and growth rates were
recorded, and haematological examinations and histopathological
studies were carried out. In particular, the lymph nodes, thymus,
spleen, and caecum were examined for distribution of pigment.
Another 2 groups of 10 rats were given basal diet or diet
containing 16% caramel colour III for 10 weeks followed by a 28-day
period during which the basal diet was fed (recovery experiment). Five
rats of each sex were killed after 7 days and the remainder after 28
days of the recovery period.
Caramel colour III depressed body-weight gain at dietary levels
greater than 1%. Total leucocyte counts were decreased in males in the
groups receiving 2, 4, and 16% caramel colour III and in females
receiving 4 and 16% caramel colour III. However, the lymphocyte/
neutrophil ratio was significantly decreased in both sexes at all dose
levels. Relative liver weights were increased at dietary levels of 2%
and higher levels of caramel colour III, and reduced spleen weights
were observed at the highest (16%) dose level. Increased relative
kidney weights were observed in both sexes at 2% and higher dietary
levels of caramel colour III. Increased caecal weights were observed
at the 16% dietary level; microscopically, pigment was observed in the
mesenteric lymph nodes of male and female rats at this dose level.
During the recovery phase, white cell counts, cell ratios, and
total numbers of lymphocytes rapidly returned to normal; recovery was
complete in both sexes by 7 days. The caecal weights had returned to
normal by 7 days and the relative liver and spleen weights returned to
normal during the recovery phase, while kidney weights partially
recovered. No details of the THI content of the caramel nor of the
pyridoxine content of diet were available (BIBRA, 1977).
Test groups of 15 male and 15 female Sprague-Dawley rats were fed
diets containing 10 or 15% caramel colour III for 4 weeks; a control
group of 20 rats of each sex received basal diet. During the course of
the study, the rats fed diets containing caramel colour III had soft
dark-coloured faeces, particularly at the highest dietary level. There
were no consistent differences in body-weight gain or food consumption
and no mortality occurred. Haematological studies were conducted prior
to feeding caramel colour III and after 2 and 4 weeks. In males, total
white cell and lymphocyte counts were unaffected by treatment at
either sampling interval or dose level. In females, total white cell
and lymphocyte numbers were significantly depressed after 4 weeks but
not at 2 weeks at both dose levels. However, significant differences
in differential white cell counts were observed in both sexes. In
males, the differential lymphocytes (%) were significantly depressed
at both dose levels and treatment intervals, and there was a
concomitant increase in segmented neutrophils. In females, the
differential counts of lymphocytes and segmented neutrophils were
similarly affected after 4 weeks.
At necropsy, increased caecal weights were observed in both sexes
and a statistically significant increase in relative weights of the
thymus in male rats fed both doses of caramel colour III was noted.
Histopathological studies were not conducted. Details of the THI
content of the caramel sample used were not available (Procter, 1977).
In a 13-week toxicity study, caramel colour III was administered
in drinking water at concentrations of 0, 4, 6, 8, or 10% to groups of
10 male and 10 female weanling Wistar rats. The caramel colour III
sample used was analysed and found to contain 78 mg/kg THI on an 'as
is' basis, 105 mg/kg THI on a solids basis. The diet fed to the rats
contained approximately 13 mg/kg pyridoxine. The general condition and
behaviour of the animals was checked at frequent intervals, individual
body weights were measured weekly, food consumption was measured (on a
cage basis) over weekly periods during the whole experimental period,
and fluid consumption was measured daily (on a cage basis). Samples of
blood for haematology were collected from the tip of the tail of all
rats initially and at days 29/30, 57/58, and 84/85. Urinalysis was
performed on individual urine samples from all animals during the last
16 hours of a 24-hour period of deprivation of food and water at day
87. Tail tip blood collected at day 87 was examined for glucose and
urea nitrogen. Blood was obtained from the aorta under ether
anaesthesia at termination (day 91 for males, 92 for females), and the
following assays performed on the plasma; alkaline phosphatase, GPT,
GOT, lactate dehydrogenase, total protein, and albumin. Pyridoxal
phosphate was measured in plasma from EDTA-treated blood samples. At
autopsy the following organs were weighed: thyroid, adrenals,
testes/ovaries, kidneys, thymus, brain, spleen, heart, liver, and
caecum (full and empty). Histological examinations were carried out on
all animals of the control and top-dose groups and, in addition to the
weighed organs, the following organs/tissues were examined: aorta,
axillary and mesenteric lymph nodes, cervix, colon, oesophagus,
stomach, duodenum, ilium, jejunum, lungs, epididymides, pituitary,
prostate, skeletal muscle, skin, sternum, pancreas, trachea, urinary
bladder, uterus, and all gross lesions.
There was a dose-related decrease in fluid consumption, decreased
urinary output of more concentrated urine, decreased food consumption,
and decreased body-weight gain in both sexes. These changes were
related to the palatability of the drinking fluid. No outstanding
differences were observed in red blood cell analyses between test
groups and controls. Total lymphocyte counts were relatively low in
all test groups of both sexes. The differences from control values
were statistically significant in all male treatment groups after
29/30 days, in the female 8% group after 29/30 days, and in the male 4
and 8% groups after 57/58 days. However, these differences did not
show a clear dose-dependent relationship and at 13 weeks there were no
significant differences among any of the treatment groups compared to
controls.
At necropsy, the relative weights of the kidneys and caecum were
increased in several groups receiving caramel colour III. The increase
in kidney weight was dose-dependent, but no treatment-related
histopathological changes were seen in any of the organs examined and
the enlargement of the kidneys was attributed to the decreased fluid
consumption by the treated rats. No effects on the relative weights of
spleen or thymus were observed and these tissues were histologically
normal (Sinkeldam et al., 1980b).
A 13-week toxicity study was conducted with caramel colour III
given in the drinking water at concentrations of 0, 5, 10, 15, or 20%
to groups of 10 male and 10 female weanling Wistar rats. The caramel
colour III sample used in this study contained 0-3 mg/kg THI on an 'as
is' basis. The diet fed to the rats contained 13.5 mg/kg pyridoxine.
The protocol used was similar to that described in the previous study
except that blood samples were collected at days 30/31, 57/58, and
80/81, and urinalysis was performed at day 85.
Water intake (fluid intake corrected for caramel colour solids
content) showed a dose-related decrease in both sexes. Food intake was
also generally lower in all treated groups. Body-weight gains of males
were decreased in a dose-related manner, while body-weight gains of
females were comparable to controls. Urine volumes were decreased in
rats treated with caramel colour III and the urines were more
concentrated in treated groups than in controls. These changes were
attributed to the decreased water intake. The urine was darker-
coloured at the 15 and 20% dose levels; however, urine composition was
essentially normal. Lymphocyte counts were in general relatively low
in all test groups in both sexes but the differences only attained
statistical significance in males of the top-dose group after 30 days
and males of all treatment groups after 57 days. No significant
differences in lymphocyte counts were observed in males of any group
after 80 days, nor in females at any dose level at any time interval.
Mean neutrophil counts were significantly increased in females
receiving 20% caramel colour III for 81 days.
Although the relative weights of the caecum, liver, brain,
kidneys, and testes were increased in several test groups, there were
no pathological changes in any of these organs. Treatment-related
microscopic changes consisting of increased numbers of macrophages
containing a yellow-brown PAS-positive pigment were found in the
mesenteric lymph nodes. These were the only treatment-related changes
noted (Sinkeldam et al., 1980a).
Groups of 10 male and 10 female weanling F344 rats were given
caramel colour III in the drinking water at concentrations of 0, 0.5,
1.0, 2.0, 4.0, or 8.0% for 4 weeks. The sample of caramel colour III
used was found to contain 70 mg/kg THI on an 'as is' basis and the NIH
07 Open Formula Mouse and Rat diet used in this study contained
17 mg/kg pyridoxine.
No differences in body-weight gain were noted for any of the test
groups, although food consumption of the males was significantly lower
than controls throughout the study. Transient decreases in lymphocyte
counts were noted among the treatment groups at the midpoint of the
study, but no significant differences between control and treated
groups were noted at 1 month. Other haematological and clinical
chemistry parameters were normal for all groups. At necropsy no
differences in organ weights were noted between treated and control
groups and there were no gross or microscopic pathological changes
related to treatment (Heidt and Rao, 1981).
Groups of 10 male and 10 female F344 rats were given caramel
colour III in the drinking water at concentrations of 0, 1.25, 2.5,
5.0, 10.0, or 20.0% for 13 weeks in order to select doses for long-
term carcinogenicity/chronic toxicity studies. Rats were given
20 ml/day of these solutions and basal diet (CRF-1, Charles River
Japan, Inc.) was available ad libitum. The caramel colour III used
contained 78 mg/kg THI on an 'as is' basis and the diet contained
11-12 mg/kg pyridoxine. During the experimental period, all animals
were observed daily and clinical signs were recorded. Body weights
were measured every other week and haematological examinations were
performed every 4 weeks in the control and 20% groups. At the end of
the study, all survivors were sacrificed for gross and microscopic
examination.
Weight gains were less in all experimental groups than in the
control group from the first experimental week, and in week 13 the
weight gains of the 1.25, 2.5, 5.0, 10.0, and 20.0% groups were 89,
94, 84, 76, and 76%, respectively, of that of controls for males and
96, 98, 80, 84, and 92%, respectively, for females. Except in the male
2.5% group and the female 1.25, 2.5, and 20.0% groups, the differences
in weight gain from the controls were significant. No haematological
changes were observed either during or at the end of the experimental
period. At necropsy, no pronounced macroscopic changes were observed
in any animals, although a few rats in the experimental groups were
very emaciated. No histological changes related to caramel colour III
administration were found in any experimental groups.
From these results, the authors concluded that 1 and 4% caramel
colour III in drinking water were the appropriate dose levels for a
carcinogenicity study (Maekawa et al., 1983).
In a 90-day toxicity study, 2 samples of caramel colour III were
used, one containing approximately 15 mg/kg THI on a solids basis
(batch A) and the other containing 295 mg/kg THI on a solids basis
(batch B). Groups of 20 male and 20 female weanling F344 rats were
given caramel colour III in drinking water at dose levels of 0, 10,
15, or 20 g/kg b.w. of batch A and 20 g/kg b.w. of batch B. The NIH 07
Open Formula Mouse and Rat diet used in this study contained more than
10 mg/kg pyridoxine. During the study, body weights and food intake
were recorded weekly. Fluid consumption was recorded 3 times per week
and concentrations of the test material were adjusted on the basis of
body weight and fluid intake to give the required dose. Haematological
analyses were performed at 2 and 6 weeks and at termination, and
clinical chemistry analyses were carried out at 6 weeks and
termination. At necropsy, all animals were examined macroscopically
and selected organ weights were determined. Histopathological
examinations were conducted on the 20 g/kg test groups and on the
control animals.
Although the findings were not always consistent, the animals
treated at the higher dose levels (15 and 20 g/kg) of batch A and
those teated with batch B generally had decreased body weights. All
treated groups had significantly decreased food and fluid intake. If
fluid intake values are corrected for solid contents of the caramel
colours, the values for water intake were markedly below those of the
controls. Because of the rather constant fluid intake of the treated
groups, it was necessary to increase progressively the caramel colour
concentration to maintain a constant intake of the caramel colour on a
body-weight basis. It is likely that the effects on body-weight gain
and food consumption were due to the reduced water intake of the rats,
reflecting the poor palatability of the drinking solution rather than
toxic effects of the caramel colour per se.
The haematology studies revealed decreased lymphocyte counts in
the male and female rats fed batch B at 2 weeks and in the male rats
fed batch B at 6 weeks. All lymphocyte values in these groups were
normal at the termination of the study. No decreases in lymphocyte
counts occurred in male or female groups fed batch A at any of the
dose levels. There were no consistent changes in clinical chemistry
values, with the exception of slightly increased values in blood urea
nitrogen in the rats treated with batch B. Urinalysis revealed
decreased urinary volume and increased specific gravity in rats of
both sexes treated with either batch of caramel colour III at 6 weeks.
At termination, these differences were only significant in male rats
in the top-dose groups (both batches). Treatment-related increases
were observed in the absolute and relative weights of the caecum (full
and empty) in animals of both sexes at all dose levels. Dose-related
increases occurred in absolute and relative kidney weights and were
considered to reflect compensatory hypertrophy as a consequence of
reduced water intake; there were no histopathological changes in the
kidneys of any of the test groups. A dose-related decrease in the
absolute weight of the thymus was observed, which reached statistical
significance in the top-dose group males with both samples of caramel
colour III and top-dose group females with batch B; this decrease was
not evident when the thymus weight was expressed relative to body
weight. Other differences in absolute and relative organ weights
appeared to be a consequence of the dose-related reduction in body
weight.
The only treatment-related microscopic changes noted were minimal
to moderate accumulation of pigment in the tissues of the intestinal
tract and mesenteric lymph nodes without pathologic alteration.
Although statistically-significant changes were identified in
some clinical and anatomical parameters, the authors did not consider
them to be toxicologically important. On this basis, the no-adverse-
effect level for ammonia caramel III was considered to be 20 g/kg b.w.
(MacKenzie, 1985).
A paired-feeding study was conducted to determine whether poor
palatability is the mechanism underlying the decreased body-weight
gains frequently noted in toxicity studies of caramel colour III. A
group of 10 male Wistar rats were fed 12% caramel colour III in the
drinking water, and similar groups were permitted a limited intake of
food or water equivalent to that consumed by the caramel colour III
group. Body-weight gain and food and fluid intake were decreased in
the group fed caramel colour III. A similar decrease in body-weight
gain was noted in the groups of rats restricted to the equivalent
intake of either food or water. The author concluded that the growth
depression observed in rats when caramel colour III is fed in the
drinking water is the result of decreased fluid and food intake. Poor
palatability of drinking fluid containing caramel colour III was the
probable cause of these changes (Sinkeldam, 1979).
Long-term studies
Rats
Four groups of 48 male and 48 female Wistar rats were given diets
containing 0, 1, 3, or 6% caramel colour III (ammonia catalysed "half
open-half closed pan" caramel; no indication about the presence of
4-methylimidazole was given) for 2 years. Food and water intake,
growth, mortality, and organ weights were measured; haematological
examinations, urinalyses, kidney function tests, and histopathological
examinations also were carried out.
A decrease in growth, which was significant in the males, was
observed at all dose levels. This effect 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 or 6% caramel. In the female
animals the lymphocytopenia was present until week 52 in these 2
groups (the 1% group was not tested).
Spleen weights were reduced in a dose-related manner. The
relative weights of the (full) caeca were clearly increased at all
dose levels. No changes were found in the pancreata of the control
animals, while in the test groups (not dose-related) a total of 10
hyperplastic changes were found. However, the number of tumours of the
pancreas showed no relation to the administration of caramel. There
was no evidence of a carcinogenic effect.
The authors concluded that a no-observed-effect level 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 with caramel colour III, a decrease
in the total number of leucocytes associated with a decrease in the
number of lymphocytes was noted. A pilot study in humans was carried
out in which 1.5 g of caramel colour III (prepared by a closed-pan
process) was ingested daily by 9 volunteers for 21 days. Total
circulating leucocytes, lymphocytes, and erythrocytes, together with
haemoglobin concentrations, were measured prior to and during the
treatment. No changes were found that could be attributed to treatment
with caramel colour III. In this experiment 6 of the subjects showed
no differences from normal in stool frequency or condition. Three
volunteers occasionally had soft stools; no control groups were used
(BIBRA, 1976).
Comments
The temporary ADI for caramel colour III was revoked at the
twenty-first meeting of the Committee due to its effects on
circulating total leucocytes and lymphocytes. Since that
time, a component of caramel colour III, 2-acetyl-4(5)-
tetrahydroxybutylimidazole (THI), has been identified and shown to
cause a depression of lymphocyte counts; the lymphocyte depression
caused by caramel colour III has been shown to be due largely, if not
solely, to this minor component. Comparison of the lymphocyte-
depressing activity of pure THI with a batch of caramel colour III
containing a known level of THI indicated that other components of
this batch had an insignificant activity. The lymphocyte depression
was largely ameliorated by dietary pyridoxine. In studies on samples
of caramel containing 10 mg/kg THI, no effects on lymphocyte counts
were observed with adequate dietary levels of pyridoxine.
Long-term studies in rats and mice indicate that caramel colour
III is not carcinogenic at dose levels of up to 4% in drinking water,
which was also the no-effect level in these long-term studies.
EVALUATION
Level causing no toxicological effect
As it was not possible to include caramel colour III at higher
levels than 4% in drinking water in long-term studies, and as the
effect of most concern, i.e. lymphopenia, could best be evaluated from
short-term studies, the Committee based its evaluation on the
no-effect level of 20 g/kg b.w./day in a 90-day study in rats using
caramel colour III which contained approximately 15 ppm THI on a
solids basis (10 ppm on an 'as is' basis).
Estimate of acceptable daily intake for man
0-200 mg/kg b.w. (0-150 mg/kg b.w. on a solids basis).
Further work or information
Desired
Analytical data to confirm that the sample on which the
evaluation is based is representative of current commercial samples.
Studies to confirm that THI is the sole component of caramel
colour III that has lymphocyte-depressing activity and to establish a
no-effect level for THI.
REFERENCES
Allen, J.A., Brooker, P.C., Birt, D.M., & McCaffrey, K.J. (1984).
Analysis of metaphase chromosomes obtained from CHO cells
cultured in vitro and treated with caramel colour III.
Unpublished report No. ITC 3B/84966 from Huntingdon Research
Centre, Huntingdon, England. Submitted to WHO by International
Technical Caramel Association.
Allen, J.A. & Proudlock, R.J. (1984). Autoradiographic assessment of
DNA repair in mammalian cells after exposure to caramel colour
III. Unpublished report from Huntingdon Research Centre,
Huntingdon, England. Submitted to WHO by International Technical
Caramel Association.
Ashoor, S.H. & Monte, W.C. (1983). Mutagenicity of commercial
caramels. Cancer Letters 18, 187-190.
BIBRA (1976). A study of the haematological effects of caramel in
human volunteers. Unpublished report No. 1/172/76 from the
British Industrial Biological Research Association, Carshalton,
Surrey, England.
Chacharonis, P. (1960). Acute and chronic toxicity studies on caramel
colours A and B. Unpublished report No. S.A. 54219 from
Scientific Associates Inc., St. Louis, MO, USA.
Chacharonis, P. (1963). Acute oral toxicity study in rats on caramel
colorings 25A-1, 30B-0, and 30F-1. Unpublished report No. S.A.
79105 from Scientific Associates Inc., St. Louis, MO, USA.
Cimino, M.C. & Brusick, D.J. (1981). Mutagenicity evaluation of ETA
48-IH caramel color in the mouse micronucleus test. Unpublished
report No. 22129 from Litton Bionetics Inc., Kensington, MD, USA.
Submitted to WHO by International Technical Caramel Association.
Evans, J.G., Butterworth, K.R., Gaunt, I.F., & Grasso, P. (1976).
Long-term toxicity study in the rat of a caramel produced by the
"half-open-half closed pan" ammonia process. Unpublished report
No. 6/1976 from the British Industrial Biological Research
Association, Carshalton, Surrey, England.
Foote, W.L., Robinson, R.F., & Davidson, R.S. (1958). Toxicity of
caramel color products. Unpublished report of Battelle Memorial
Institute, Columbus, OH, USA.
Galloway, S.M. & Brusick, D.J. (1981a). Mutagenicity evaluation of
ETA-48-IH in an in vitro cytogenetic assay measuring chromosome
aberration frequencies in Chinese hamster ovary (CHO) cells.
Unpublished report No. 20990 from Litton Bionetics Inc.,
Kensington, MD, USA. Submitted to WHO by International Technical
Caramel Association.
Galloway, S.M. & Brusick, D.J. (1981b). Mutagenicity evaluation of
ETA-48-IH in an in vitro cytogenetic assay measuring chromosome
aberration frequencies in Chinese hamster ovary (CHO) cells. Part
II. Unpublished report No. 20990 from Litton Bionetics Inc.,
Kensington, MD, USA. Submitted to WHO by International Technical
Caramel Association.
Gaunt, I.F., Lloyd, A.G., Grasso, P., Gangolli, S.P., & Butterworth,
K.R. (1975). Toxicological investigations of caramels. I. A
short-term study in the rat with two caramels produced by
variations of the ammonia process. Unpublished report No. 14 from
the British Industrial Biological Research Association,
Carshalton, Surrey, England.
Hagiwara, A., Shibata, M., Kurata, Y., Seki, K., Furushima, S., & Ito,
N. (1983). Long-term toxicity and carcinogenicity test of
ammonia-process caramel colouring given to B6C3F1 mice in the
drinking water. Fd. Chem. Toxicol. 21, 701-706.
Haldi, J., & Wynn, W. (1951). A study to determine whether or not
caramel has any harmful physiological effect. I. Unpublished
report from Emory University, Atlanta, GA, USA.
Heidt, M. & Rao, G.N. (1981). Subchronic toxicity study of ammonia
caramel color type AC2 in rats. Unpublished report No. 80036 from
Raltech Scientific Services, Inc., Madison, WI, USA. Submitted to
WHO by International Technical Caramel Association.
Ishidate, M. Jr. & Yoshikawa, K. (1980). Chromosome aberration tests
with Chinese hamster cells in vitro with and without metabolic
activation - a comparative study on mutagens and carcinogens.
Arch. Toxicol. Suppl. 4, 41-44.
Jagannath, D.R. & Brusick, D. (1978a). Mutagenicity evaluation of ETA
4-10, ETA 4-11, ETA 4-15 in the Ames Salmonella/microsome plate
test. Unpublished report No. 20838 from Litton Bionetics Inc.,
Kensington, MD, USA. Submitted to WHO by International Technical
Caramel Association.
Jagannath, D.R. & Brusick, D. (1978b). Mutagenicity evaluation of ETA
4-8, ETA 4-9, ETA 4-12, ETA 4-13, ETA 4-14 in the Ames
Salmonella/microsome plate test. Unpublished report No. 20838
from Litton Bionetics Inc., Kensington, MD, USA. Submitted to WHO
by International Technical Caramel Association.
Jensen, N.J., Willumsen, D., & Knudsen, I. (1983). Mutagenic activity
at different stages of an industrial ammonia caramel process
detected in Salmonella typhimurium TA 100 following pre-
incubation. Fd. Chem. Toxicol. 21, 527-530.
Kawachi, T., Yahagi, T., Kada, T., Tazima, Y., Ishidate, M., Sasaki,
M., & Sugiyama, T. (1980). Cooperative programme on short-term
assays for carcinogenicity in Japan. In Molecular and Cellular
Aspects of Carcinogen Screening Tests, IARC Scientific
Publication No. 27, 323-330. International Agency for Research
on Cancer, Lyon, France.
Kawana, K., Akema, R., Nakaoka, T., Ikeda, H., Takimoto, T., &
Kawauchi, S. (1980). Studies on mutagenicities of natural food
additives. II. Mutagenicities and antibacterial activities of
caramels. Eisei Kagaku 26, 259-263.
Kroplien, U. (1984). Letter dated 17 December 1984 to International
Technical Caramel Association, submitted to WHO.
Kroplien, U., Rosdorfer, J., van der Greef, J., Long, R.C. Jr., &
Goldstein, J.H. (1985). 2-Acetyl-4(5)-(tetrahydroxybutyl)-
imidazole: Detection in commercial caramel colour III and
preparation by a model browning reaction. J. Org. Chem. 50,
1131-1133.
MacKenzie, K.M. (1985). 90-day toxicity study of caramel color
(ammonia process) in rats. Vols. I & II. Unpublished report No.
6154-107 from Hazleton Laboratories America, Inc., Madison, WI,
USA. Submitted to WHO by International Technical Caramel
Association.
Maekawa, A., Ogiu, T., Matsuoka, C., Onodera, H., Furuta, K.,
Tanigawa, H., Hayashi, Y., & Odashima, S. (1983). Carcinogenicity
study of ammonia-process caramel in F344 rats. Fd. Chem.
Toxicol. 21, 237-244.
Morgareidge, K. (1974). Teratologic evaluation of FDA 71-82 (caramel,
bakers and confectioners) in mice, rats and rabbits. Unpublished
report No. PB 234-870 from Food and Drug Research Laboratories
Inc., Waverly, NY; USA.
Nishie, K., Waiss, A.C., & Keyl, A.C. (1969). Toxicity of
methylimidazoles. Toxicol. Appl. Pharmacol. 14, 301-307.
Nishie, K., Waiss, A.C., & Keyl, A.C. (1970). Pharmacology of alkyl
and hydroxyalklpyrazines. Toxicol. Appl. Pharmacol. 17, 1.
Procter, B.G. (1976). A preliminary evaluation of the potential
toxicological effects of ammonia caramel in mice. Unpublished
report No. 5705 from Bio-Research Laboratories Ltd., Pointe
Claire, Quebec, Canada.
Procter, B.G. (1977). A preliminary evaluation of the potential
toxicological effects of ammonia caramel (U.S. origin) in rats.
Unpublished report No. 5617 from Bio-Research Laboratories Ltd.,
Pointe Claire, Quebec, Canada. Submitted to WHO by International
Technical Caramel Association.
Procter, B., Berry, G., & Chappel, C.I. (1976). A toxicological
evaluation of various caramels fed to albino rats. Unpublished
report No. 4244 from Bio-Research Laboratories Ltd., Pointe
Claire, Quebec, Canada.
Richold, M. & Jones, E. (1980). Ames metabolic activation test to
assess the potential mutagenic effect of ETA-48-1H. Unpublished
report No. FDC 8/80358 from Huntingdon Research Centre,
Huntingdon, England. Submitted to WHO by International Technical
Caramel Association.
Richold, M., Jones, E., & Fenner, L.A. (1984). Ames metabolic
activation test to assess the potential mutagenic effect of
caramel colour III. Unpublished report No. ITC 1B/84709 from
Huntingdon Research Centre, Huntingdon, England. Submitted to WHO
by International Technical Caramel Association.
Sharratt, M. (1971). Unpublished report.
Sinkeldam, E.J. (1979). Paired feeding test in rats with caramel
(AC3) in drinking water. Unpublished report No. R 6157 from
Centraal Instituut voor Voedingsonderzoek (CIVO/TNO), Zeist, The
Netherlands. Submitted to WHO by International Technical Caramel
Association.
Sinkeldam, E.J. (1981). Quantitative relationship between dietary
pyridoxine content and lymphocyte counts in rats fed ammonia
caramel. Unpublished report No. V81.390/211704 from Centraal
Instituut voor Voedingsonderzoek (CIVO/TNO), Zeist, The
Netherlands. Submitted to WHO by International Technical Caramel
Association.
Sinkeldam, E.J. (1982a). Quantitive relationship between dietary
pyridoxine content and lymphocyte counts in mature rats fed
ammonia caramel. Unpublished report No. V82.102/220102 from
Centraal Instituut voor Voedingsonderzoek (CIVO/TNO), Zeist, The
Netherlands. Submitted to WHO by International Technical Caramel
Association.
Sinkeldam, E.J. (1982b). Short-term (7 day) bioassay in rats with
three dose levels of 2-acetyl-4(5)-tetrahydroxybutylimidazole
(13-A-82). Unpublished report No. V82.291/221153 from Centraal
Instituut voor Voedingsonderzoek (CIVO/TNO), Zeist, The
Netherlands. Submitted to WHO by International Technical Caramel
Association.
Sinkeldam, E.J., Bruyntjes, J.P., & Kuper, C.F. (1980a). Sub-chronic
(13-week) oral toxicity study with a modified ammonia caramel
(ETA-26-1) in rats. Unpublished report No. R 6483 from Centraal
Instituut voor Voedingsonderzoek (CIVO/TNO), Zeist, The
Netherlands. Submitted to WHO by International Technical Caramel
Association.
Sinkeldam, E.J., Roverts, W.G., & Kuper, C.F. (1980b). Sub-chronic
(13-week) oral toxicity study with ammonia caramel (AC2) in
rats. Unpublished report No. R 6121 from Centraal Instituut voor
Voedingsonderzoek (CIVO/TNO), Zeist, The Netherlands. Submitted
to WHO by International Technical Caramel Association.
Sinkeldam, E.J., de Groot, A.P., van den Berg, H., & Chappel, C.I.
(1984). The effect of vitamin B6 on the number of lymphocytes in
blood of rats fed caramel colour (ammonia process). Unpublished
report submitted to WHO by International Technical Caramel
Association.
Sinkeldam, E.J. & van der Heyden, C.A. (1976a). Short-term feeding
test with three types of caramels in albino rats. Unpublished
report No. R 4789 from Centraal Instituut voor Voedingsonderzoek
(CIVO/TNO), Zeist, The Netherlands.
Sinkeldam, E.J. & van der Heyden, C.A. (1976b). Short-term (10 week)
feeding study in rats with three different ammonia caramels.
Unpublished report No. R 5120 from Centraal Instituut voor
Voedingsonderzoek (CIVO/TNO), Zeist, The Netherlands.
CARAMEL COLOUR IV
EXPLANATION
The report of the twenty-fourth meeting of the Committee (Annex
1, reference 53) drew attention to the need for adequate
specifications for caramel colour IV and for a long-term study of
carcinogenicity. The temporary ADI of 0-100 mg/kg b.w. was extended
pending the results of long-term toxicity studies.
BIOLOGICAL DATA
Biochemical Aspects
Absorption, distribution, and excretion
Pigmentation of tissues of the lymphoreticular system,
particularly of the mesenteric lymph nodes, has been a frequent
observation in rats fed high levels of caramel colour. A study was
undertaken to determine the distribution of the colour component of
caramel colour in rats and to determine whether accumulation of
caramel colour was the cause of pigmentation in the mesenteric lymph
nodes.
Experimental batches of unlabelled and 14C-labelled caramel
colour IV were prepared and a concentrate of the higher molecular-
weight colour components was made by ultrafiltration. The absorption,
tissue distribution, and excretion of this fraction was studied
in male F344 rats after a single oral gavage of 2.5 g/kg b.w.
14C-Labelled material was administered to naive animals and to
animals which had received 2.5 g/kg b.w. unlabelled material in
drinking water daily for 13 days prior to administration of
14C-labelled material.
Differences between the results in naive and pretreated animals
were small. Most of the colour components were not absorbed, but
instead were excreted in the faeces, mainly within 48 hours. By 96
hours after dosing, 99.7-102.4% of the administered dose was excreted
in the faeces, with only 1-2% in urine and insignificant amounts
(< 0.1%) as 14CO2 in expired air.
Groups of 4 rats were killed at intervals of 4, 8, 12, 24, and 96
hours after receiving the single oral dose of 14C-labelled material,
and radioactivity was measured in the following tissues/organs; blood,
brain, heart, lungs, liver, kidneys, spleen, thymus, mesenteric and
cervical lymph nodes, gastrointestinal tract (contents and tissues),
and carcass. Most of the radioactivity was located in the
gastrointestinal tract, and only low levels of radioactivity were
found in blood and tissues. The specific activity in the thymus,
mesenteric lymph nodes, spleen, kidneys, and liver exceeded that in
blood, but with the exception of the mesenteric lymph nodes, the
radioactivity was cleared rapidly over the 96-hour study period. With
the exception of the gastrointestinal tract, the highest tissue levels
attained, in the liver and kidneys, never exceeded 0.02% and 0.01% of
the administered dose.
The results demonstrate that, after administration of large doses
of the coloured components of caramel colour IV, only a small fraction
was absorbed, distributed in lymphoreticular tissue, and eventually
excreted in the urine; retention by the mesenteric lymph nodes
appeared to account for the pigmentation observed in this tissue
(Selim, 1982).
Toxicological studies
Special studies on carcinogenicity (See also long-term studies)
Mice
A carcinogenicity study of caramel colour IV was performed on
B6C3F1 mice using 5 groups of 50 male and 50 female mice in each
group. Two groups of each sex served as controls and the 3 treatment
groups received caramel colour IV in the drinking water at dose levels
of 2.5, 5.0, or 10.0 g/kg b.w./day for 104 weeks. All animals were
observed twice daily and palpated for tissue masses weekly from week
27. Body weights and food intake were recorded weekly, and fluid
intake was measured on the first, third, fifth, and seventh days of
the first 12 weeks and over a 48-hour period each week thereafter.
A complete necropsy was performed on all animals dying on test or
sacrificed in a moribund condition, and on all survivors at
termination. Histology (haematoxylin and eosin staining) was performed
on all gross lesions and on the following organs/tissues of all
animals: adrenals, brain (three sections), bone, bone marrow,
epididymis, oesophagus, eyes, gall bladder, heart, intestine
(duodenum, ileum, caecum and colon), kidneys, liver, lymph nodes
(cervical, mesenteric, and thoracic), mammary glands, ovaries,
pancreas, parathyroid, pituitary, prostate, salivary gland, sciatic
nerve, seminal vesicle, skin, spinal cord, spleen, stomach, testes,
thymus, thyroids, trachea, urinary bladder, and uterus.
Four animals were found in the wrong cages during week 15 and had
probably been misplaced 9 weeks earlier. These animals were removed
from the study during week 31 and were not included in subsequent
examinations.
Although during the course of this study there were sporadic
significant differences in mean body weights of treated male and
female groups compared to controls, caramel-colour feeding did not
have consistent effects on body weights or body-weight gains in either
sex. Males of the 10 g/kg b.w.-dose group had lower mean food
consumption than the control groups for 67 of the 104 weeks and males
receiving 2.5 g/kg b.w. had lower mean food consumption for 21 of the
104 weeks. There were no consistent differences in food intake of
males at the 5 g/kg b.w. dose level nor of any of the females.
Decreased fluid intake was noted, particularly in males at the higher
dose levels; mean fluid intakes of treated females were usually equal
to or only slightly lower than controls. There were no treatment-
related differences in survival rates, which at termination were
65-75% for the male groups and 67-79% for the female groups. At
necropsy, there were dose- and/or treatment-related effects on the
gastrointestinal tract and mesenteric lymph nodes, including dark
gastrointestinal contents, staining of the mucosa, and diffusely red
and congested mesenteric lymph nodes. These changes were not
considered to be toxicologically important and there were no other
changes of toxicological significance. There was no evidence of
treatment-related neoplastic lesions in any organs (MacKenzie, 1985b).
Rats
A long-term toxicity and carcinogenicity study of caramel colour
IV was conducted on F344 rats. In the carcinogenicity portion of the
study, 5 groups of 50 male and 50 female weanling rats were selected
randomly; 2 groups of each sex served as controls and the 3 treatment
groups received caramel colour IV in drinking water at dose levels of
2.5, 5.0, or 10 g/kg b.w./day for 24 months. In the chronic toxicity
portion of the study, groups of 25 male and 25 female rats received
caramel colour IV in drinking water at doses of 0, 2.5, 5.0, 7.5, or
10 g/kg b.w./day for 1 year. The following parameters were monitored:
mortality, clinical observations including ophthalmic changes, body
weight, and food and fluid consumption. In the chronic toxicity
portion clinical chemistry and haematological studies were done at
intervals of 6 and 12 months and necropsies were performed at 12
months.
In the carcinogenicity portion of the study, a complete
necropsy was performed on all animals dying on test or sacrificed in
moribund condition, and on all survivors at termination. Histology
(haematoxylin and eosin staining) was performed on all gross lesions
and on the following organs/tissues of all animals: adrenals, brain
(three sections), bone, bone marrow, epididymis, oesophagus, eyes,
heart, intestine (duodenum, ileum, caecum, and colon), kidneys, liver,
lung, lymph nodes (cervical, mesenteric, and thoracic), mammary gland,
ovaries, pancreas, parathyroid, pituitary, prostate, salivary gland,
sciatic nerve, seminal vesicle, skin, spinal cord, spleen, stomach,
testes, thymus, thyroid, trachea, urinary bladder, and uterus.
The feeding of caramel colour IV did not affect survival in
either the chronic toxicity or carcinogenicity sections of this study
and, other than dark-stained and soft faeces, there were no treatment-
related antemortem observations. During the course of both studies
body weights were reduced for both males and females at the 5 and
10 g/kg b.w. dose levels. These effects on body weights were
correlated with reduced water and food consumption at these dose
levels and reflect the reduced palatability of the drinking fluid.
Clinical chemistry and haematological studies at 6 and 12 months
in the chronic toxicity study did not reveal changes of toxicological
concern. At 6 months, serum concentrations of blood urea nitrogen and
creatinine were reduced in male groups treated with 5.0, 7.5, and
10 g/kg b.w. and in female groups treated with 7.5 and 10 g/kg b.w.
caramel colour. Similar changes were noted at 12 months. Creatinine
levels were within the normal range for the F344 rat, whereas blood
urea nitrogen levels were slightly outside this range. Decreased
levels of serum total protein, albumin, and globulin were also noted
at the 6-month sampling, particularly in male rats. These changes,
which were less marked at 12 months, were not accompanied by any
pathology in the liver or kidneys and were not considered to be of
toxicological importance.
The urinalysis studies revealed generally reduced urine volume
and increased specific gravity in both sexes.
At necropsy the changes noted were characteristic of the feeding
of high levels of caramel colour, which consisted primarily of brown
staining of the gastrointestinal tract and mesenteric lymph nodes and
caecal enlargement. Increased kidney weights were noted in both sexes
in animals fed caramel colour IV; no histologic alterations were
present that could be associated with the increased renal weight,
which the authors considered to be related to the water imbalance in
these animals.
Microscopic examination of the tissues of these animals did not
reveal specific toxicological changes. Pigmentation in the
gastrointestinal tract and mesenteric lymph nodes was observed. There
was no evidence of reactive hyperplasia to the pigment in the
mesenteric lymph nodes of the gastrointestinal tract.
In the chronic portion of the toxicity study, although
statistically significant changes were noted in some parameters, they
were not considered to be toxicologically important. The highest dose
fed, 10 g/kg b.w., was considered to be the no-adverse-effect level.
In the carcinogenicity portion of the study, the observations
were generally similar to those in the chronic portion. Survival at 24
months ranged from 64-68% for males and 82-92% for females. Random
variations in both benign and malignant neoplasms typical of the F344
strain and this age of animal were observed; however, there were no
treatment-related differences. The authors concluded that the feeding
of caramel colour IV at doses up to 10 g/kg b.w. for 24 months did not
induce neoplastic changes or non-neoplastic changes of toxicological
importance (MacKenzie, 1985a).
Special studies on mutagenicity
Caramel colour IV was evaluated for genetic activity in a series
of in vitro microbial assays with and without metabolic activation.
Salmonella typhimurium (strains TA1535, TA1537, and TA1538) and
Saccharomyces cerevisiae were used. Caramel colour IV was not
genetically active under the test conditions employed in this study
(Brusick, 1974).
Caramel colour IV was evaluated for mutagenicity using the Ames
Salmonella/microsome plate test and the Saccharomyces/microsome
plate test. The samples tested were blends of 3 samples of caramel
colour IV (low colour intensity) and 3 samples of caramel colour IV
(high colour intensity). The Salmonella test organisms used were
TA98, TA100, TA1535, TA1537, and TA1538. Tests were conducted directly
or in the presence of liver microsomal enzyme preparations from
Araclor-induced rats. Tests were conducted over a range of
concentrations from 1-50 mg/plate. No signs of genetic activity were
observed with any of the samples of caramel colour IV tested using
either the Salmonella or Saccharomyces test organisms (Jagannath &
Brusick, 1978a).
The clastogenicity of caramel colour IV (low colour intensity)
was evaluated in an in vitro cytogenetic assay using cultured
Chinese hamster ovary cells without metabolic activation. No increase
in chromosome aberrations was observed at concentrations of test
material between 5 µg/ml and 5 mg/ml. In the same test, sodium
ascorbate produced a positive response at 2 mg/ml (Galloway & Brusick,
1981a).
The clastogenicity of caramel colour IV (high colour intensity)
was evaluated in an in vitro cytogenetic assay using Chinese hamster
ovary cells without metabolic activation. No increase in chromosome
aberrations was observed at concentrations of test material between
5 µg/ml and 5 mg/ml. In the same test, sodium ascorbate produced a
positive response at 2 mg/ml (Galloway & Brusick, 1981b).
Two samples of caramel colour IV of medium and high colour
intensity were assayed for mutagenic activity in the Ames test using
Salmonella strains TA98 and TA100 in the absence or presence of rat
hepatic S-9 fraction. Neither sample showed mutagenic activity under
the conditions of the test (Ashoor & Monte, 1983).
Thirteen commercial caramel colours (not identified) were
examined for mutagenicity in the Ames test using Salmonella strains
TA98 and TA100, with and without metabolic activation, and for
DNA-damage effects on E. coli (Wild/pol A-; Wild/rec A-). None of
the caramel-colour samples tested showed mutagenic activity in these
tests (Kawana et al., 1980).
Five samples of commercial caramel colour were tested for
mutagenic activity against Salmonella strains TA98 and TA100 in the
Ames test. All samples were reported to show equivocal results with
strain TA100, and 2 were equivocal with TA98 (Kawachi et al., 1980).
Five samples of caramel colour were tested in a chromosome
aberration test in a cultured cell line of Chinese hamster lung
fibroblasts, in the presence or absence of hepatic S-9 fraction. Ames
tests were also conducted using Salmonella strains TA98, TA100, and
TA1537, with or without metabolic activation and with a 20-minute
pre-incubation step. All samples of caramel colour were designated as
positive in the Ames assay and in the chromosome aberration test
(aberrations were detected in 20% of metaphase cells) (Ishidate &
Yoshikawa, 1980).
The same series of 5 caramel-colour samples as used in the above
studies were tested for mutagenicity in the Ames test using
Salmonella strains TA98, TA100, TA1535, TA1537, and TA1538 and
Saccharomyces strain D4. Assays were performed both in the presence
and absence of hepatic microsomal S-9 fraction from Araclor-induced
rats in the conventional plate assay and after a pre-incubation step
in strains TA100 and TA1535. The results of the mutagenicity assays
were negative under all the test conditions and over a concentration
range of 1-50 mg/plate for strain TA100 and 1-20 mg/plate for the
other strains (Jagannath & Brusick, 1978b).
Special studies on reproduction
Fifteen male and female Wistar rats were given 0 or 10% caramel
colour IV solution as their sole fluid source until day 100 and were
then mated. Animals of the F1-generation (25 males and 25 females)
were weaned and again given 0 or 10% caramel solution until day 100.
There were no adverse effects with regard to the number of litters
born and the number of pups/litter. No influence on haematology,
growth, food consumption, gross pathology, or histopathology of the
F1-generation at 100 days of age was observed (Haldi & Wynn, 1951).
Six different samples of caramel colour IV (3 single-strength
(SS) and 3 double-strength (DS) products), each containing a different
level of 4-methylimidazole (between 200 and 850 ppm) were tested in a
reproduction study in rats. Two control diets were used, an unmodified
stock diet and the stock diet supplemented with starch and cellulose.
The various test diets are shown in the following diagramme.
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 colour
IV + 202b 5.6 11.5 17.6
SS caramel colour
IV + 400 11.5
SS caramel colour
IV + 600 11.5
DSc caramel colour
IV + 350 2.3 4.5 6.8
DS caramel colour
IV + 639 4.5
DS caramel colour
IV + 852 4.5
a SS=Single stength
b Quantity of 4-methylimidazole in ppm
c DS=Double strength
Twelve groups of 10 male and 20 female weanling Wistar rats were
allocated to the above dietary groups and, at week 12, the rats were
mated in sub-groups of 5 males and 10 females. After a 3-week mating
period, the females were caged individually.
At weaning age of the F1 generation, 40 males and 40 females
were selected from as many different litters as possible within each
caramel-colour group and continued on the same diet as the parent
generation. Ten males and 10 females of each group were sacrificed
after one year (see Sinkeldam et al., 1975, under short-term
toxicity studies); the remaining 30 males and 30 females of each group
were fed the test diets for 2 years (see Sinkeldam et al., 1976, under
long-term toxicity studies). After weaning the F1 litters, the dams
were killed and the implantation sites were counted.
No consistent, dose-related effects on growth of the F0 animals
were noted. No adverse effects were seen on female fertility, litter
size, average weight and growth of the pups, or number of implantation
sites or sex ratio of the young. In one group (10% SS caramel colour
IV + 600 ppm 4-methylimidazole) there was a slight increase in
mortality at birth. No teratogenic effects were found (Til & Spanjers,
1973).
A range-finding reproduction study was conducted on F344 rats.
Five groups of 12 male and 12 female mature rats were given caramel
colour IV at concentrations of 0, 10, 15, 20, or 25% in drinking water
for 21 days prior to mating and throughout mating, gestation, and
lactation. Animals of the F0 generation were killed when animals from
the F1 generation were weaned. At weaning, 2 pups/sex/litter were
randomly selected from litters that had a minimum of 2 males and 2
females at day 21 and treatment of these animals was continued for 13
weeks post-weaning. Haematology and clinical chemistry were performed
on days 45/46 and at termination. Complete gross post-mortem
examinations of both F0 and F1 animals were performed at sacrifice.
Selected organs (caecum, spleen, thymus, liver, kidneys, heart,
adrenals, and gonads from animals of the F0 generation were weighed
at autopsy.
Although the dose levels of caramel colour IV in this study were
very high (ranging from 8-28 g/kg b.w.), no specific toxic effects
were observed. All F0-generation animals survived the duration of the
study, but 3 F1-generation animals were killed accidentally while
sampling for clinical chemistry studies at day 45. In the 20- and
25%-dose groups of both generations there was a higher incidence of
soft stools than in the controls, and all animals of the F0
generation showed slight to statistically significant, dose-related
decreases in body weight. Dose-related depression of body-weight gain
was also noted in animals of the F1 generation.
Mating, pregnancy, and fertility rates were comparable for all
groups, but the number of implantation sites and of pups alive at days
0, 4, and 21 of lactation in the 20%-dose groups was significantly
lower than control values. Litter size was decreased at the 15-, 20-
and 25%-dose levels. Pups in the 25%-dose group showed a markedly
higher incidence of alopecia and arched spine than controls, and a
generalized poor condition during the last 7 days of suckling. No
significant haematological changes were observed at 45 or 90 days
post-weaning, except that prothrombin time of the F1 females in the
15 and 25% groups were significantly greater than controls at day 45.
Blood urea nitrogen values were lower than controls at 45 and 90 days
but other clinical chemistry values were normal.
At necropsy, dose-related increases in absolute and relative
weights of the liver, kidneys, and caecum (full and empty) were
observed from animals in the 15%- and higher-dose groups. The only
gross treatment-related morphological changes reported were
brown/black or green colouration of the contents and mucous membrane
of the lower gut and mesenteric lymph nodes (Tierney, 1980).
Special studies on teratogenicity
Teratogenicity tests were performed with caramel colour IV on
mice, rats, and rabbits. The doses employed were 0, 16, 74.3, 345, and
1600 mg/kg b.w. in all 3 species.
Mice
Caramel colour IV was administered by gavage to groups of 19-22
pregnant albino CD1 mice at the doses above, beginning on day 6 and
continuing through day 15 of gestation. Body weights were recorded and
all animals were observed daily for changes in appearance and
behaviour. On day 17 all dams were subjected to Caesarean section
under surgical anaesthesia and the numbers of implantation sites,
resorption sites, live and dead foetuses, and body weights of live
pups were recorded. All foetuses were examined grossly for external
congenital abnormalities, one-third of the foetuses from each litter
underwent visceral examination (Wilson technique), and the remaining
two-thirds were cleared, stained with Alizarin Red, and examined for
skeletal defects.
The number of abnormalities seen in either soft or skeletal
tissues did not differ from the number occurring spontaneously in
sham-treated controls (Morgareidge, 1974).
Rats
Caramel colour IV was administered by gavage to groups of 21-24
pregnant Wistar rats at the dose levels above, beginning on day 6 and
continuing daily through day 15 of gestation. Body weights were
recorded and all animals were observed daily for changes in appearance
and behaviour. On day 20 dams were subjected to Caesarean section
under surgical anaesthesia and the number of implantation sites,
resorption sites, live and dead foetuses, and body weights of live
pups were recorded. All foetuses were examined for gross, visceral,
and skeletal abnormalities as in the mouse experiment.
No clearly discernible effects on nidation or on maternal or
foetal survival were observed. The number of abnormalities seen in the
test groups did not differ from the number occurring spontaneously in
sham-treated controls (Morgareidge, 1974).
Rabbits
Caramel colour IV was administered by gavage to groups of 11-12
pregnant Dutch-belted female rabbits at the doses above beginning on
day 6 and continuing daily through day 18 of pregnancy. Body weights
were recorded and all animals were observed daily for changes in
appearance and behaviour. The does were subjected to Caesarian section
under surgical anaesthesia on day 29 and the numbers of corpora lutea,
implantation sites, resorption sites, and live and dead foetuses were
recorded.
Body weights of the live pups were also recorded. All foetuses
were examined grossly for the presence of external congenital
abnormalities. The live foetuses from each litter were then placed in
an incubator for 24 hours for evaluation of neonatal survival.
Surviving pups were sacrificed and all pups examined for visceral
abnormalities (by dissection). All foetuses were then cleared with
potassium hydroxide, stained with Alizarin Red, and examined for
skeletal defects.
No clearly discernible effects on nidation or on maternal or
foetal survival were observed. The number of abnormalities seen in
either soft or skeletal tissues of the test groups did not differ from
the number occurring spontaneously in sham-treated controls
(Morgareidge, 1974).
Special study on haematology
Rats
A study was conducted in rats to determine whether haematological
changes were associated with the feeding of high dietary
concentrations of caramel colour IV. Sixty-six male rats, mean initial
body weights approximately 173 g, were fed caramel colour IV in the
diet for 28 days. Sixteen rats were assigned to a control group and 10
rats/group were assigned to levels of 16 or 22% caramel colour IV
(high colour intensity) and 34 or 47% caramel colour IV (low colour
intensity). Caramel colour III was fed at a 4% level to a positive
control group. The parameters observed were body weight, food
consumption, haematology (total and differential leucocyte counts),
and terminal necropsy with organ weights (caecum - full and empty).
Rats were bled from the retro-orbital sinus on days 16, 9, and 2
before treatment and again on days 7, 14, and 28 during treatment.
Feeding of these high dietary levels of caramel colour IV led to
reduced rates of body-weight gain. The animals fed caramel colour III
gained weight at a rate similar to the controls. Animals receiving
caramel colour III had progressively lower relative lymphocyte counts
commencing 1 week after treatment and increasing in severity with
duration of treatment. This pattern of decreased relative lymphocyte
counts was not seen with either sample of caramel colour IV and no
significant decreases in lymphocyte counts were observed at any dose
level of this caramel colour. Caecal weights were increased in all
treatment groups (BIBRA, 1978).
Acute toxicity
No information available.
Short-term studies
Mice
Groups of 10 B6C3F1 mice of each sex were given concentrations
of 0, 10, 15, 20, or 30% caramel colour IV in drinking water for 4
weeks. The intake of caramel colour IV expressed in g/kg b.w./day was
more than twice the percentage concentration in the drinking water. At
28 days the body weights of male animals at the 30%-dose level were
significantly decreased compared to the controls, and transient
depressions of body weights were noted at the highest-dose level in
females. No significant depressions in body-weight gain were noted at
the lower-dose levels in either male or female mice. Fluid consumption
was depressed throughout the study among all treatment groups.
However, these changes were not consistent with time or dosage. There
were no statistically significant differences in food consumption
between treated animals and controls. At necropsy, the only treatment-
related effect reported was enlargement of the caecum (Tierney, 1979).
Rats
Groups of 5 rats received either 10 or 20% caramel colour IV
solution (equivalent to about 10 or 20 g/kg b.w./day) as their 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 pathology,
or histopathology (Haldi & Wynn, 1951).
Six groups of 5 male and 5 female weanling rats received 0 or 10%
caramel colour IV solution as their sole fluid source for 100, 200, or
300 days. No adverse effects were noted regarding growth, food and
fluid intake, haemotology, gross pathology, or histopathology (Haldi &
Wynn, 1951).
Groups of 16 male and 16 female rats received either 0 or 10%
caramel colour IV solution for 100 days and groups of 5 rats received
20% caramel solution for 100 days. At the lower test level, there were
no observable abnormalities as regards growth, food consumption,
haematology, gross pathology, or histopathology. Only growth and
haematology were examined at the higher test level, and the results
for both parameters were found to be normal (Haldi & Wynn, 1951).
Groups of 5 male and 5 female rats were given 1 ml/kg b.w. of
concentrated caramel colour for 21 days. Some diarrhoea was induced in
all animals, but no other abnormalities were noted. Gross pathology
and histopathology revealed no significant changes due to
administration of the test compound (Foote et al., 1958).
Three groups of 20 male and female rats received either 0 or
11-14 g/kg b.w. of caramel colour IV solutions for 100 days. Growth
and food intake did not differ significantly between test and control
animals. Gross pathology and histopathology showed no abnormal
findings related to administration of the test compound (Haldi & Wynn,
1958).
Four groups of 10 male and 10 female Sprague-Dawley rats received
0, 0.1, 1.0 or 10% caramel colour IV in their diet for 12 weeks. No
adverse effects were noted on growth, food consumption, urinalysis,
haematology, gross pathology, or histopathology that were 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 IV in their diet for 3 months. Weight gain was normal
in all groups. Food consumption, haematology, and urinalysis were
comparable in all groups. Gross pathology and histopathology showed no
test-related adverse findings (Chacharonis, 1960).
Four groups of 10 male and 10 female Sprague-Dawley rats received
0, 5, 10, or 20% caramel colour IV (low colour intensity) or caramel
colour IV (high colour intensity) in their diet for 90 days. In
addition, a paired-feeding study involving 5 male rats in 2 groups was
run for 23 days (one sample was at the 20% level), and there were no
differences 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% levels due to unpalatability of the test diet. No other adverse
findings were noted in growth, behaviour, mortality, haematology,
urinalysis, gross pathology, organ weights, or histopathology (Kay and
Calandra, 1962a; 1962b).
Four groups of 10 male and 10 female Sprague-Dawley rats received
either 0 or 10% of 3 different caramel colours (one of which was a
sample of caramel colour IV) in their diet for 90 days. Weight gains
showed a slight reduction compared with controls but food consumption
was normal for all groups. No abnormalities were noted with regard to
haematology, urinalysis, gross pathology, or histopathology
(Chacharonis, 1963).
Four groups of 15 male and 15 female rats received 0, 5, 10, or
20% caramel colour IV 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
pathology, or histopathology (Oser, 1963).
Four groups of 10 male and 10 female rats received 0, 0.015, 0.3,
or 3.0% caramel colour IV in their diet for 90 days. No differences
between test and control animals were noted regarding body weight,
food consumption, haematology, urinalysis, gross pathology, or
histopathology (Nees, 1964).
Four groups of rats received 0, 4, 8, or 16% caramel colour IV
in their diet for 3 months. No convulsions or other behavioural
abnormalities or signs of neurological damage were seen. No
macroscopic or microscopic pathological abnormalities were found in
the central nervous system (Sharratt, 1971).
Three groups of 20 female Wistar rats received stock diet to
which 0, 10, or 20% caramel colour IV containing 202 ppm 4-
methylimidazole was added. During the second week of the experiment
these levels of caramel colour IV were increased to 15 and 25% and in
week 7, the levels were increased to 25 and 30% caramel colour IV. The
diets were administered for 16 weeks followed by a 4-week recovery
period.
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 after 4 weeks of feeding caramel colour IV.
After the recovery period of 4 weeks the increases had disappeared.
The relative weight of the thymus was not affected.
Gross examination at autopsy after 16 weeks of feeding caramel
clour IV revealed a dose-related, brown-greenish discolouration of the
mesenteric lymph nodes in all test animals of the highest-dose level.
After the recovery period of 4 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 4 weeks. These results failed to confirm
the decreased leucocyte counts that were observed in females fed 5 to
15% caramel colour IV in a 1-year feeding study (Sinkeldam & van der
Heyden, 1975).
A 10-week feeding study with 18 groups of 10 male and 10 female
Wistar rats was carried out with 3 caramel colours, including caramel
colour IV (low colour intensity) and caramel colour IV (high colour
intensity). The dietary concentrations were 0, 1.25, 2.5, 5, 10, and
15% caramel colour IV (low colour intensity) and 0, 0.5, 1, 2, 4, and
6% caramel colour IV (high colour intensity).
Both caramel colours caused loose stools at the highest-dose
levels, although body-weight gains were not affected. Leucocyte
(lymphocyte) counts were not affected in the animals fed samples of
caramel colour IV at any of the dose levels. Only slight indications
of caecal enlargement were observed. Minimal amounts of pigment were
observed in the mesenteric lymph nodes of rats fed 2.5% and higher of
the low colour intensity sample, and 1% and higher of the high colour
intensity sample (Sinkeldam and van der Heyden, 1976).
In a 10-week feeding study, 17 groups of 15 male and 15 female
Sprague-Dawley rats were given various caramel colours that included
caramel colour IV (low colour intensity) and caramel colour IV (high
colour intensity). The dose levels fed were 1.25, 2.5, 5, 10, and 15%
for caramel colour IV (low colour intensity) and 0.5, 1, 2, 4, and 6%
caramel colour IV (high colour intensity). Two control groups were
used.
In the animals fed caramel colour IV (low colour intensity) at
the 15% level, the faeces became soft within 2 weeks. The water
content of the faeces from these animals was higher than that of the
controls, as was the water content of faeces from rats fed 6% caramel
colour IV (high colour intensity). Body-weight gains were slightly
decreased in male, but not female, rats fed both samples of caramel
colour.
There were no significant changes in total white cell or
lymphocyte counts in animals fed either sample of caramel colour IV.
No macroscopic or microscopic evidence of abnormal pigmentation of the
mesenteric lymph nodes was found in any group of either sample of
caramel colour IV. Caecal weights were generally increased in all test
groups fed both samples of caramel colour IV (Procter et al., 1976).
Groups of weanling Wistar rats were given caramel colour IV (low
colour intensity) or caramel colour IV (high colour intensity) at
concentrations of 0, 0.5, 1.0, 2.0, 4.0, or 16.0% in the diet for 10
weeks. Each group contained 15 male or 15 female rats except the
control group (60 animals of each sex) and the 16%-dose group
(10 animals of each sex). Food intake and growth were recorded and
haematological studies were carried out. Lymph nodes, thymus, spleen,
and caecum were examined histologically for distribution of pigment.
An additional 3 groups of 10 rats of each sex were given basal
diet or diet containing 16% each of the caramel colours for 10 weeks.
At the end of this period all the rats received basal diets.
Haematological studies were performed on these animals at 3, 7, 14,
and 28 days. Five rats of each sex were killed after 7 days and the
remainder after 28 days of feeding basal diet (recovery experiment).
Decreased body-weight gains were noted in animals of both sexes
fed 16% caramel colour IV (high colour intensity). No decreases were
observed in the groups fed caramel colour IV (low colour intensity).
Food intake was not consistently altered in any of the groups fed
either sample of caramel colour IV. Occasionally, values for total
leucocyte counts were significantly higher or lower than controls in
the groups fed both samples of caramel colour IV; however, these
changes were not consistent in direction and they were not dose-
related. There were no consistent or dose-related differences in
lymphocyte counts between the groups fed caramel colour IV and the
controls. Liver weights were significantly increased in the group fed
16% caramel colour IV (high colour intensity). Increased relative
kidney weights were observed in the groups fed 2, 4, and 16% caramel
colour IV (high colour intensity) and 16% caramel colour IV (low
colour intensity), although no histological changes were observed.
Increased caecal weights were seen only at the 16% feeding level for
both caramel colours. At necropsy pigmentation of the lymph nodes
was seen at the 16% feeding level of both caramel colours.
Microscopically, pigmentation was observed in the mesenteric lymph
nodes in the males and females in the groups fed 16% caramel colour IV
(high colour intensity). Relative weights of the liver and kidneys and
caecal weights returned to normal during the recovery period (BIBRA,
1977).
Five groups of 30 male and 30 female weanling F344 rats were
given caramel colour IV in drinking water at concentrations which
provided intakes of 0, 15, 20, 25, or 30 g/kg b.w./day for 13 weeks.
After 43 days, 10 animals of each sex from each group were randomly
selected for collection of data on urinalysis, haematology, and
clinical chemistry, followed by sacrifice and necropsy; similar
examinations were performed on survivors at termination. At interim
and terminal sacrifice, a detailed necropsy was performed on all
animals and a complete histopathological examination was performed on
controls and animals in the top-dose group.
Throughout the study, rats given caramel colour IV produced dark-
coloured, soft or sticky, liquid and/or odorous, faeces which stained
and caused alopecia of the perianal area, most noticeably at the
higher-dose levels. Dose-related decreases in food intake, water
consumption (after correction for caramel content), and body-weight
gains were observed and were attributed to the poor palatability of
the drinking solutions.
Caramel colour IV at the dose levels tested did not significantly
affect haematological values at interim or terminal examinations. All
treatment groups of both sexes had significantly-reduced blood urea
nitrogen and alkaline phosphatase levels at both 45 and 90 days. Total
serum protein values of both sexes in the treatment groups were lower
than controls at 90, but not at 45, days. These effects may be due to
reduced food intake and growth retardation.
Treated rats had reduced urine volume and increased urine
specific gravity, protein, ketones, and acidity, which were associated
with decreased water consumption. Increased kidney weights were
observed at necropsy. Treatment-related decreases in thymus and spleen
weights and increased caecal size, with staining of the mucosa of the
caecum and colon, were noted. Accumulation of yellowish-tan pigment
occurred in macrophages of the mesenteric lymph nodes. No treatment-
related histopathological changes were observed in any organs at the
highest-dose level (Heidt & Rao, 1980).
A 1-year rat toxicity study was conducted as a continuation of
the reproduction study described earlier (Til & Spanjers, 1973). It
involved the interim sacrifice of one-fourth of the animals utilized
in the 2-year toxicity study described below.
Wistar rats (10 males or 10 females in each group) were selected
from the first litter of parents that were given diets containing 6
samples of caramel colour IV, 3 of which were low colour intensity and
3 of which were high colour intensity. The dose levels employed were
5, 10, and 15% caramel colour IV (low colour intensity) and 2, 4, and
6% caramel colour IV (high colour intensity). Two additional samples
of caramel colour IV (low colour intensity) were tested at 10% and 2
additional samples of caramel colour IV (high colour intensity) were
tested at 4% in the diet. The animals were sacrificed after a feeding
period of 1 year.
No adverse effects on behaviour, growth, food intake, mortality,
liver and kidney function tests, urine composition, or organ weights
were observed. Haematological indices showed a slight dose-related
decrease in total leucocyte counts in females fed one sample of
caramel colour IV (low colour intensity) which contained 202 mg/kg
methylimidazole. In males, no effect was noted and in a subsequent
experiment in which the same sample of caramel colour IV 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 attributed to the feeding of caramel colour IV
consisted of increased accumulation of a yellow-brown pigment and
pigment-laden macrophages in the mesenteric lymph nodes of males and
females in all caramel colour IV groups. Inflammatory or degenerative
changes of the lymphoid tissue were not found (Sinkeldam et al.,
1975).
Dogs
Four groups of 3 male and 3 female adult beagle dogs received 0,
6, 12.5, or 25% caramel colour IV in their diet 5 days per week for 90
days. No significant adverse effects on growth, behaviour, food
consumption, mortality, liver function, kidney function, haematology,
urinalysis, gross pathology, or histopathology were noted (Kay and
Calandra, 1962c).
Long-term toxicity studies
Rats
Six samples of caramel colour were tested in a long-term toxicity
study, 3 of which were caramel colour IV (low colour intensity) and 3
of which were caramel colour IV (high colour intensity). Each group
consisted of 40 male or 40 female weanling Wistar rats, except the
control group which had double this number of animals. Animals were
selected from the first litter of parents fed diets containing the
various caramel colours from weaning age (see reproduction studies,
Til & Spanjers, 1973). The dose levels tested were 5, 10, or 15%
caramel colour IV (low colour intensity) and 2, 4, or 6% caramel
colour IV (high colour intensity). Two additional samples of caramel
colour IV (low colour intensity) were tested at 10% and 2 additional
samples of caramel colour IV (high colour intensity) were tested at 4%
in the stock diet.
Observations were made of general appearance, behaviour,
mortality, growth, food intake, haematological factors, and clinical
chemistry of blood and urine. After about 14 months mortality
attributed to intercurrent disease was observed in both control and
treated groups. Approximately three-quarters of the animals died or
were killed before the experiment was terminated at week 104. Organs
of animals that died or were killed were weighed and extensive
histopathological examinations were carried out. 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 surviving rats
of all groups.
No clinical changes were observed in this study except for
slightly decreased haemoglobin and haematocrit values at weeks 78 and
98 in males fed caramel colour IV (high colour intensity). Leucocyte
counts were decreased in females fed 10 and 15% of one sample of
caramel colour IV (low colour intensity) at weeks 13 and 52, but these
changes were not consistent and decreases in lymphocyte counts were
not reported. At autopsy, an increased incidence of greenish
discoloured mesenteric lymph nodes was observed in most groups fed
high levels of caramel colour. Microscopically, an increased pigment-
phagocytosis in the mesenteric lymph nodes in all test groups (except
the group fed 2% caramel colour IV (high colour intensity)) 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 caramel colour IV. An increase in the incidence of
neoplastic lesions in the different groups was not found (Sinkeldam
et al., 1976).
A long-term toxicity and carcinogenicity study of caramel colour
IV was conducted using F344 rats (MacKenzie, 1985a). Details are given
above (see special studies on carcinogenicity).
Observations in man
Tolerance studies of caramel colour IV (low colour intensity) and
caramel colour IV (high colour intensity) were conducted in human
volunteers. The subjects, 10 men and 10 women, ingested caramel colour
once daily in simulated soft drinks over 3 test periods of 21 days
each separated by 7-day rest intervals. The test doses were 6 g/day
during the first test period, 12 g/day during the second period, and
18 g/day during the third test period.
Haematological, clinical chemical, and routine urinary parameters
were studied at the beginning of each ingestion period, after 10 days
of ingestion, and at the end of each ingestion period. Most individual
values for haemoglobin, haemotocrit, RBC, corrected sedimentation
rate, WBC, and differentials (neutrophils, basophils, eosinophils,
monocytes, and lymphocytes) were found to be within normal limits.
There were a few instances of values outside the normal range,
indicating mild neutropenia and mild lymphocytosis, but these were not
consistent and were unrelated to the ingestion of caramel colour IV.
On the other hand, caramel ingestion was associated with an increased
frequency of bowel movements and softening or increased liquidity of
faeces (Marier et al., 1977a; 1977b).
Comments
The pigmentation of mesenteric lymph nodes and caecal enlargement
were considered to be non-specific effects that are of no
toxicological significance. The carcinogenicity studies required by
the twenty-fourth meeting of the Committee (Annex 1, reference 53)
have been conducted in rats and mice, and no treatment-related
neoplastic changes were observed. The material used in these studies
conformed to recent specifications. The committee based its evaluation
on the no-effect level in these long-term/carcinogenicity studies, to
which (in view of the ancillary human data in which no adverse effects
other than laxation were observed) a safety factor of 50 was applied.
EVALUATION
Level causing no toxicological effect
Mouse: 10 g/kg b.w./day in drinking water
Rat: 10 g/kg b.w./day in drinking water
Man: No adverse effects other than laxation at levels up to
18 g/day.
Estimate of acceptable daily intake for man
0-200 mg/kg b.w. (0-150 mg/kg b.w. on a solids basis).
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Industrial Bio-test Laboratories, Inc., Northbrook, IL, USA.
Kay, J.H. & Calandra, J.C. (1962b). Ninety-day subacute oral toxicity
of caramel coloring (coded Sample B) - albino rats. Unpublished
report from Industrial Bio-test Laboratories, Inc., Northbrook,
IL, USA.
Kay, J.H. & Calandra, J.C. (1962c). Subacute oral toxicity of caramel
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Industrial Bio-test Laboratories, Inc., Northbrook, IL, USA.
MacKenzie, K.M. (1985a). Carcinogenicity and chronic toxicity study of
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Laboratories America, Inc., Madison, WI, USA. Submitted to WHO by
International Technical Caramel Association.
MacKenzie, K.M. (1985b). Carcinogenicity study of caramel color
(sulfite ammonia process) type SAC2 in mice. Vols. I-VI.
Unpublished study No. 81185 from Hazleton Laboratories America,
Inc., Madison, WI, USA. Submitted to WHO by International
Technical Caramel Association.
Marier, G., Berry, G., & Orr, J.M. (1977a) Tolerance study of single
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project No. 5612, report No. 2, from Bio-Research Laboratories
Ltd., Pointe Claire, Quebec, Canada.
Marier, G., Berry, G., & Orr, J.M. (1977b) Tolerance study of double
strength ammonia sulfite caramel in human volunteers. Unpublished
project No. 5711, report No. 2, from Bio-Research Laboratories
Ltd., Pointe Claire, Quebec, Canada.
Morgareidge, K. (1974). Teratologic evaluation of FDA 71-83 (caramel,
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NY, USA.
Nees, P.O. (1964). Toxicological feeding study of caramel E.
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Madison, WI, USA.
Oser, B.L. (1963). Toxicological feeding study of "acid-proof"
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Laboratories, Inc., Waverly, NY, USA.
Prier, R.F. (1960). The toxicity of double strength acid proof caramel
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from Wisconsin Alumni Research Foundation, Madison, WI, USA.
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Selim, S. (1982). Single and repeat oral dose pharmacokinetic and
distribution studies of caramel color concentrate in the rat.
Unpublished study No. IT-59r from Primate Research Institute,
Holloman Air Force Base, NM, USA. Submitted to WHO by
International Technical Caramel Association.
Sharratt, M. (1971). Unpublished report.
Sinkeldam, E.J. & van der Heyden, C.A. (1975). Short-term feeding
study with caramel SS 202 in albino rats. Unpublished report No.
R 4777 from Centraal Instituut voor Voedingsonderzoek (CIVO/TNO),
Zeist, The Netherlands.
Sinkeldam, E.J. & van der Heyden, C.A. (1976). Short-term (10 week)
feeding study in rats with three different ammonia caramels.
Unpublished report No. R 5120 from Centraal Instituut voor
Voedingsonderzoek (CIVO/TNO), Zeist, The Netherlands.
Sinkeldam, E.J., van der Heyden, C.A., & Beems, R.B. (1976). Chronic
(two year) feeding study in rats with six different ammonia
caramels. Unpublished report No. R 4961 from Centraal Instituut
voor Voedingsonderzoek (CIVO/TNO), Zeist, The Netherlands.
Sinkeldam, E.J., Willems, M.I., & van der Heyden, C.A. (1975). One
year feeding study in rats with six different ammonia caramels.
Unpublished report No. R 4767 from Centraal Instituut voor
Voedingsonderzoek (CIVO/TNO), Zeist, The Netherlands.
Tierney, W.J. (1979). A four-week dose range-finding study of caramel
colour no. 3, sample 3-1, in mice. Unpublished project
No. 79-2380 from Bio/dynamics Inc., East Millstone, NJ, USA.
Submitted to WHO by International Technical Caramel Association.
Tierney, W.J. (1980). An in-utero range-finding study of caramel
colour no. 3, sample 3-1, in rats. Unpublished project
No. 79-2405 from Bio/ynamics Inc., East Millstone, NJ, USA.
Submitted to WHO by International Technical Caramel Association.
Til, H.P. & Spanjers, M. (1973). Reproduction study in rats with six
different ammonia caramels. Unpublished report No. R 4068 from
Centraal Instituut voor Voedingsonderzoek (CIVO/TNO), Zeist, The
Netherlands.
See Also:
Toxicological Abbreviations