INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
WORLD HEALTH ORGANIZATION
TOXICOLOGICAL EVALUATION OF SOME
FOOD COLOURS, ENZYMES, FLAVOUR
ENHANCERS, THICKENING AGENTS, AND
CERTAIN FOOD ADDITIVES
WHO FOOD ADDITIVES SERIES 6
The evaluations contained in this publication were prepared by the
Joint FAO/WHO Expert Committee on Food Additives which met in Rome,
4-13 June 19741
World Health Organization Geneva 1975
1 Eighteenth Report of the Joint FAO/WHO Expert Committee on
Food Additives, Wld Hlth Org. techn. Rep. Ser., 1974, No. 557.
FAO Nutrition Meetings Report Series, 1974, No. 54.
CARAMEL COLOUR (AMMONIA PROCESS)
These compounds have been evaluated for acceptable daily intake
by the Joint FAO/WHO Expert Committee on Food Additives (see Annex I,
Ref. No. 19) in 1971.
Since the previous evaluation additional data have become
available and are summarized and discussed in the following monograph.
The previously published monograph has been expanded and is reproduced
in its entirety below.
Caramel refers to a large number of poorly defined and complex
products formed from various carbohydrates generally by heating with
any of a wide range of acids, bases and salts, under varying
conditions of temperature and pressure. It might be argued that
caramel can be considered as a natural constituent of the diet as it
can be formed when certain foods are cooked or when sucrose is heated.
Toxicological discrimination is unwarranted between such caramel and
caramels produced commercially from food-grade carbohydrates, with the
exception of caramels prepared by processes using ammonia or ammonium
The production of violent hysteria and convulsions in cattle and
sheep fed ammoniated sugar-containing feed supplements (nitrogen
content 4-6%), at 6-25% of their rations led to the discovery of the
presence of about 20% of pyrazines and 10% of imidazoles in these
ammonium-treated molasses (WHO, 1970). 4-methylimidazole has been
shown to be the most likely toxic component being a convulsant to
rabbits, mice and chicks at oral doses of 360 mg/kg bw (Nishie et al.,
1969). The pyrazines, on the other hand, are mild CNS depressants and
weak anticonvulsants (Heyns, 1971). Analysis of food grade caramel
colours, however, showed that only 0.002-0.02% of 4-methylimidazole
is present in commercial products (Heyns, 1970). Commercial caramel
colours of undefined origin contain 50-500 ppm 4-methylimidazole
(Heyns, 1971) while other examinations have shown ranges of
100-700 ppm (Battelle Memorial Institute, 1971). It has been shown
that the yields of imidazole compounds increased linearly with the
increment of molar ratio of ammonia to glucose (Komoto, 1962). Further
analyses for 4-methylimidazole were being carried out on a large
variety of caramel colours (Nishie et al., 1971). Generally caramel
colours contain 50% digestible carbohydrate, 25% non-digestible
carbohydrate and 25% of melanoidins also found in roasted coffee,
broiled meats and baked cereal products.
In groups of two to four rats, the absorption of the colour-
giving components of caramel was determined by faecal extraction.
Recoveries varied widely for the 10 or 20% caramel solutions examined
despite pretreatment 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).
Special studies on reproduction
Fifteen male and 15 female rats were given 0 or 10% caramel
solution as their sole fluid source until day 100 and were then mated.
The F1 generation was weaned and given again 0 or 10% caramel
solution until day 100. It showed no adverse effects as regards
litter number, haematology, growth, food consumption, gross and
histopathology (Haldi & Wynn, 1951). A further reproduction study
including teratology is planned (CIVO, 1972).
Animal Route (mg/kg bw) Reference
Rat Oral > 2.3 ml approx. or = 1 900 Foote et al., 1958
Oral > 25 ml approx. or = 17 500 Chacharonis, 1960
Oral > 30 ml approx. or = 20 400 Chacharonis, 1963
No abnormalities were detected after observation of animals for
14 days following administration of 12 different caramel colour
products mostly based on ammonia or ammonium sulfate catalysts (Foote
et al., 1956; Chacharonis, 1960; Chacharonis, 1963). A single dose of
up to 10 g/kg bw in mice and 15 g/kg in rabbits of caramels produced
by the ammonia catalyser closed pan process or sodium hydroxide
process did not cause convulsions or other signs of distress
Groups of five male and five female rats were given 1 ml/kg bw of
concentrated caramel colour for 21 days. Some diarrhoea was induced
in all animals but no other abnormalities were noted. Gross and
histopathology revealed no significant changes due to administration
of the test compound (Foote et al., 1958).
Groups of five rats received either 10 or 20% caramel solution
equivalent to about 10 or 20 g/kg bw as sole source of fluid for 127
days. Only dark faeces and very mild diarrhoea were noted. No adverse
effects were noted regarding general health, body weight, food and
fluid consumption, haematology, gross and histopathology (Haldi &
Six groups of five male and five female weanling rats received 0
or 10% caramel solution as their sole fluid source for 100, 200 or 300
days respectively. No adverse effects were noted regarding growth,
food and fluid intake, haematology, gross and histopathology (Haldi &
Groups of 16 male and 16 female rats received either 0 or 10%
caramel solution for 100 days and groups of five rats received 20%
caramel solution for 100 days. At the lower test level there were no
observable abnormalities as regards growth, food consumption,
haematology, gross and histopathology. Only growth and haematology
were examined at the higher test level (Haldi & Wynn, 1951).
Three groups of 20 male and female rats received either 0 or
11-14 g/kg bw of caramel solutions for 100 days. Growth and food
intake did not differ significantly between test and control animals.
Gross and histopathology showed no abnormal findings related to
administration of the test compound (Haldi, 1958).
Four groups of 10 male and 10 female rats received 0, 0.1, 1.0
and 10% of caramel colour in their diet for 12 weeks. No adverse
effects were noted on growth, food consumption, urinalysis,
haematology, gross and histopathology related to administration
of the caramel colour (Prier, 1960).
Groups of 10 male and 10 female rats received 0, 5, or 10 g/kg
caramel colour in their diet for three months. Weight gain was normal
in all groups. Food consumption, haematology and urinalysis were
comparable. Gross and histopathology showed no test-related adverse
findings (Chacharonis, 1960).
Four groups of 10 male and 10 female rats received 0, 5, 10 and
20% of two different caramel colours in their diet for 90 days. In
addition, a paired feeding study involving five male rats in two
groups was run for 23 days with one sample at the 20% level, and there
was no difference in the rate of growth. The only effects attributable
to treatment were a mild depression in growth of male rats at the 10
and 20% level due to impalatability of the test diet. No other adverse
findings were noted in growth, behaviour, mortality, haematology,
urinalysis, gross pathology, organ weights, and histopathology (Kay &
Four groups of 10 male and 10 female rats received either 0 or
10% of three different caramel colours in their diet for 90 days.
Weight gains showed slight reduction compared with controls but food
consumption was normal for all groups. No abnormalities were noted
regarding haematology, urinalysis, gross and histopathology
Four groups of 15 male and 15 female rats received 0, 5, 10 and
20% of caramel colour in their diet for 90 days. No adverse effects
were noted on appearance, behaviour, survival, body weights, food
intake, haematology, blood chemistry, urinalysis, organ weights, gross
and histopathology (Oser, 1963).
Four groups of 10 male and 10 female rats received 0, 0.015, 0.3
and 3.0% of caramel colour in their diet for 90 days. No differences
between test and control animals were noted regarding body weight,
food consumption, haematology, urinalysis, gross or histopathology
Four groups of rats received 0, 4, 8 and 16% caramel colour in
their diet for three months. No convulsions or other behaviour
abnormality or signs of neurological damage were seen. No macroscopic
or microscopic pathological abnormalities were found in the CNS. Other
results are still to come (Sharratt, 1971).
Four groups of three male and throe female adult beagles received
0, 6, 12.5 and 25% of caramel colour in their diet five days per week
for 90 days. No significant adverse effects were noted due to the test
compound on growth behaviour, food consumption, mortality, liver
function, kidney function, haematology, urine analysis, gross and
histopathology (Kay & Calandra, 1962).
Studies on 10 groups of 40 male and 40 female rats are in
progress extending over two years and using caramels of varying
4-methylimidazole content at different dietary levels from 2 to 10%
No formal studies are available but in the course of a
reproduction study using 25 pairs of 1 male and 1 female rat given 0
or 0.8 g/kg bw caramel colour in their drinking fluid as part of a
beverage tested, several pairs survived for two years or longer. No
deleterious effects on growth were noted. The data are rather
incomplete (Bachmann et al., 1946).
A large number of caramel colours has been tested in short-term
studies in rats and one variety has been tested in dogs. An extensive
programme of long-term and reproduction studies and embryotoxicity
including teratogenicity is under way. The acute and short-term
studies considered reveal that high levels of intake have no adverse
effects on the CNS. Therefore the acute neurological effects produced
by high doses of 4-methylimidazole would not appear to be of major
concern when caramel colours containing small amounts of this
contaminant are used in food. The analytical evidence suggests the
presence of 4-methylimidazole in the range of 50-700 ppm in caramel
colours depending upon the process of manufacture; 200 ppm is taken as
an average low value for 4-methylimidazole content. Since the effects
of chronic ingestion of 4-methylimidazole are unknown the above
mentioned long-term studies as well as more adequate reproduction,
embryotoxicity including teratology studies have been initiated on
caramel colours produced by the ammonia-ammonium sulfate process.
Level causing no toxicological effect
Rat: 20% (200 000 ppm) in the diet equivalent to 10 000 mg/kg bw.
Estimate of acceptable daily intake for man
0-100 mg/kg bw*,**
FURTHER WORK OR INFORMATION
Required before June 1978
Results of long-term and reproduction studies on caramel colours
prepared by the ammonia or ammonium sulfate process containing several
levels of 4-methylimidazole.
** Based on a product having a colour intensity of 20 000 EBC units,
containing not more than 200 ppm of 4-methylimidazole.
Battelle Memorial Institute (1971) Unpublished report dated 4 May 1971
Bachmann, G. et al. (1946) J. Nutr., 32, 85
Chacharonis, P. (1960) Unpublished report No. S.A. 54219 of Scientific
Chacharonis, P. (1963) Unpublished report No. S.A. 79105 of Scientific
CIVO, 1972, Unpublished report submitted to WHO
Foote, W. L., Robinson, R. F. & Davidson, R. S. (1958) Unpublished
report of Battelle Memorial Institute
Haldi, J. & Wynn, W. (1951) Unpublished report of Emory University
Haldi, J. (1958) Unpublished report of Emory University
Heyns, K. (1970) Unpublished report of Technical Caramel Committee
Heyns, K. (1971) Unpublished summary report
Kay, J. H. & Calandra, J. C. (1962) Unpublished report of Industrial
Bio-test Laboratories Inc.
Komoto, M. (1962) J. Agric. Chem., 36, 305
Neese, P.O. (1964) Unpublished report of Wisconsin Alumni Research
Nishie, K., Waiss, A. C. & Keyl, A. C. (1969) Toxicol. Appl.
Pharmacol., 14, 301
Nishie, K., Waiss, A. C. & Keyl, A. C. (1971) Toxicol. Appl.
Pharmacol., 17, 1
Oser, B. L. (1963) Unpublished report No. 83911 of Food and Drug
Prier, R. F. (1960) Unpublished report No. 9070599 of Wisconsin Alumni
Sharratt, M. (1971) Unpublished report