WORLD HEALTH ORGANIZATION
WHO Food Additives Series 1972, No. 1
TOXICOLOGICAL EVALUATION OF SOME
ENZYMES, MODIFIED STARCHES AND
CERTAIN OTHER SUBSTANCES
The evaluations contained in this publication were prepared by the
Joint FAO/WHO Expert Committee on Food Additives which met in Rome,
16-24 June 19711
World Health Organization
1 Fifteenth Report of the Joint FAO/WHO Expert Committee on Food
Additives, Wld Hlth Org. techn. Rep. Ser., 1972, No. 488; FAO
Nutrition Meetings Report Series, 1972, No. 50.
The monographs contained in the present volume are also issued by the
Food and Agriculture Organization of the United Nations, Rome, as FAO
Nutrition Meetings Report Series, No. 50A
(c) FAO and WHO 1972
CARAMEL COLOURS (AMMONIA PROCESS)
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
per cent.), at 6-25 per cent. of their rations led to the discovery of
the presence of about 20 per cent. of pyrazines and 10 per cent. 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 body-weight (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 per cent. 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
per cent. digestible carbohydrate, 25 per cent. non-digestible
carbohydrate and 25 per cent. of melanoidins also found in roasted
coffee, broiled meats and baked cereal products.
In groups of 2 to 4 rats, the absorption of the colour-giving
components of caramel was determined by faecal extraction. Recoveries
varied widely for the 10 or 20 per cent. 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).
Animal Route LD50 Reference
rat oral > 2.3 ml = 1 900 Foote et al., 1958
oral > 25 ml = 17 500 Chacharonis, 1960
oral > 30 ml = 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 sulphate catalysts (Foote et al.,
1956; Chacharonis, 1960; Chacharonis, 1963). A single dose of up to
10 g/kg body-weight in mice and 15 g/kg in rabbits of caramels
produced by the ammonia catalyser closed pan process or sodium
hydroxide process did not cause convulsions or other signs of distress
Groups of 5 male and 5 female rats were given 1 ml/kg bodyweight 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 5 rats received either 10 or 20 per cent. caramel solution
equivalent to about 10 or 20 g/kg body-weight as sole source of fluid
for 127 days. Only dark faeces and very mild diarrhoea were noted.
No adverse effects were noted regarding general health, body-weight,
food and fluid consumption, haematology, gross and histopathology
(Haldi & Wynn, 1951).
Six groups of 5 male and 5 female weanling rats received 0 or 10 per
cent. 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 per cent.
caramel solution for 100 days and groups of 5 rats received 20 per
cent. caramel solution for 100 days. At the lower test level there
were no observable adnormalities 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 body-weight of caramel solutions for 100 days. Growth and
food intake did not differ significantly between test and control
animals. Gross and histopathology showed no abnormal findings related
to administration of the test compound (Haldi, 1958).
Four groups of 10 male and 10 female rats received 0, 0.1, 1.0 and 10
per cent. 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 3 months. Weight gain was normal in all
groups. Food consumption, haematology and urinalysis were comparable.
Gross and histopathology showed no test-related adverse findings
Four groups of 10 male and 10 female rats received 0, 5, 10 and 20 per
cent. of two different caramel colours in their diet for 90 days. In
addition, a paired feeding study involving 5 male rats in two groups
was run for 23 days with one sample at the 20 per cent. 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 per cent. 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 & Calandra, 1962).
Four groups of 10 male and 10 female rats received either 0 or 10 per
cent. of 3 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 per
cent. of caramel colour in their diet for 90 days. No adverse effects
were noted on appearance, behaviour, survival, bodyweights, 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 per cent. 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 (Neese, 1964).
Four groups of rats received 0, 4, 8 and 16 per cent. caramel colour
in their diet for 3 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 3 male and 3 female adult beagles received 0, 6, 12.5
and 25 per cent. of caramel colour in their diet 5 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).
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
body-weight caramel colour in their drinking fluid as part of a
beverage tested, several pairs survived for 2 years or longer. No
deleterious effects on growth were noted. The data are rather
incomplete (Bachmann et al., 1946).
Fifteen male and 15 female rats were given 0 or 10 per cent. 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 per cent.
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 large variety of caramel colours has been tested in short-term
studies in rats and one variety has been tested in dogs. No
satisfactory long-term or multigeneration studies are available. The
acute and short-term studies considered reveal that at high levels of
intake there is no evidence of any convulsions or other adverse
neurological effects or of histopathological damage. Therefore, the
acute neurological effects produced by high doses of 4-methylimidazole
would not appear to be of major concern when caramel colours
containing only 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, long-term studies on caramel colours
produced by the ammonium sulphate process are required.
Level causing no significant toxicological effect in the rat
Twenty per cent. (200 000 ppm) in the diet equivalent to 10 000 mg/kg
Estimate of acceptable daily intake for man mg/kg body-weight
Temporary acceptance1 0-100
Further work required by 1974
Long-term studies on caramel colours prepared by the ammonia or
ammonium sulphate process containing several levels of
Battelle Memorial Institute (1971) Unpublished report dated 4.5.71
Bachmann, G,, Haldi, J,, Wynn. W. & Ensor, C. (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
FAO/WHO (1970) Wld Hlth Org. techn. Rep. Ser., 445
Foote, W. L., Robinson, R. F. & Davidson, R. S. (1958) Unpublished
report of Battelle Memorial Institute
Haldi, J. & Wynn, W. (1951) Unpublished report of Emery University
Haidi, 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
1 Single strength colour equivalents containing not more than 200 ppm
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