INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY WORLD HEALTH ORGANIZATION SUMMARY OF TOXICOLOGICAL DATA OF CERTAIN FOOD ADDITIVES WHO FOOD ADDITIVES SERIES NO. 12 The data contained in this document were examined by the Joint FAO/WHO Expert Committee on Food Additives* Geneva, 18-27 April 1977 Food and Agriculture Organization of the United Nations World Health Organization * Twenty-first Report of the Joint FAO/WHO Expert Committee on Food Additives, Geneva, 1977, WHO Technical Report Series No. 617 CARAMEL COLOUR (AMMONIA AND AMMONIA-SULFITE PROCESS) EVALUATION FOR ACCEPTABLE DAILY INTAKE BIOLOGICAL DATA BIOCHEMICAL ASPECTS Caramel refers to a large number of poorly defined and complex products formed from various carbohydrates generally by heating with any of a wide range of acids, bases and salts, under varying conditions of temperature and pressure. It might be argued that caramel can be considered as a natural constituent of the diet as it can be formed when certain foods are cooked or when sucrose is heated. Toxicological discrimination is unwarranted between such caramel and caramels produced commercially from food-grade carbohydrates, with the exception of caramels prepared by processes using ammonia or ammonium salts. The production of violent hysteria and convulsions in cattle and sheep fed ammoniated sugar-containing feed supplements (nitrogen content 4-6%), at 6-25% of their rations led to the discovery of the presence of about 20% of pyrazines and 10% of imidazoles in these ammonium-treated molasses. 4-methylimidazole has been shown to be the most likely toxic component being a convulsant to rabbits, mice and chicks at oral doses of 360 mg/kg body weight (Nishie et al., 1969). The pyrazines, on the other hand, are mild CNS depressants and weak anticonvulsants (Heyns, 1970). Analysis of food grade caramel colours, however, showed that only 0.002-0.02% of 4-methylimidazole is present in commercial products (Heyns, 1970). Commercial caramel colours of undefined origin contain 50-500 ppm 4-methylimidazole (Heyns, 1971) while other examinations have shown ranges of 100-700 ppm (Battelle Memorial Institute, 1971). It has been shown that the yields of imidazole compounds increased linearly with the increment of molar ratio of ammonia to glucose (Komoto, 1962). Further analyses for 4-methylimidazole were being carried out on a large variety of caramel colours (Nishie et al., 1970). Generally caramel colours contain 50% digestible carbohydrate, 25% non-digestible carbohydrate and 25% of melanoidins also found in roasted coffee, broiled meats and baked cereal products. In groups of two to four rats, the absorption of the colour- giving components of caramel was determined by faecal extraction. Recoveries varied widely for the 10 or 20% caramel solutions examined despite pre-treatment for 100 days before testing. About one-third of the colour-giving components appeared to be absorbed but no conclusions could be drawn regarding the absorption of colourless components (Haldi and Wynn, 1951). TOXICOLOGICAL STUDIES Special studies on reproduction Fifteen male and 15 female rats were given 0 or 10% ammonia- sulfite caramel solution as their sole fluid source until day 100 and were then mated. The F1-generation (25 males and 25 females) was weaned and given again 0 or 10% caramel solution until day 100. It shows no adverse effects as regards number of litters born and number of pups/litter. No influence on haematology, growth, food consumption, gross and histopathology of the F1-generation at an age of 100 days was found (Haldi and Wynn, 1951). Six different ammonia-sulfite caramels (three single strength (SS) and three double strength (DS) products) each containing a different level of 4-methylimidazole (between 200 and 850 ppm) were tested in a reproduction study with rats. Two control diets were used viz. an unmodified stock diet and the stock diet supplemented with starch and cellulose. The various test diets enable to examine the effects of 5, 10 and 15% of a single strength caramel, and of 2.4 and 6% of a double strength caramel and of 10% single strength and 4% double strength caramels each with three different contents of 4-methylimidazole (see Table 1). Twelve groups of 30 rats (a Wistar strain), 10 males and 20 females per group were used. At week 12 all rats were mated in groups of five males and ten females to produce one litter. At weaning age of the young 40 males and 40 females were selected from as many different litters as possible of each caramel group and continued on the same diet as their parents. Ten males and ten females of each group are intended to be sacrificed after one year (see short- term studies), the remaining 30 males and 30 females of each group are intended for completing a two-year feeding period (see long-term studies). After weaning the first litters, the mothers were killed to count the implantation sites. No influence on growth of the parents was noted. After the breeding no adverse effects on the number of young per litter, average weight and growth of the young, mortality (except in the group with 10% SS + 600 ppm methylimidazol, no significant increase was found), ratio males/females and number of implantation sites was found. No teratogenic effects were found (Til and Spanjers, 1973). TABLE 1. COMPOSITION OF THE TEST DIET (IN g) Group 1 2 3 4 5 6 7 8 9 10 11 12 Caramel in % 5 10 15 10 10 2 4 6 4 4 0 0 stock diet 100 100 100 100 100 100 100 100 100 100 100 100 wheat starch 3.8 1.9 - 1.9 1.9 4.9 4.1 3.4 4.1 4.1 5.8 - cellulose 4.2 2.2 - 2.2 2.2 5.4 4.6 3.8 4.6 4.6 6.2 - SSa caramel (ammonia-sulfite process) + 202b 5.6 11.5 17.6 SS caramel (ammonia-sulfite process) + 400 11.5 SS caramel (ammonia-sulfite process) + 600 11.5 DSc caramel (ammonia-sulfite process) + 350 2.3 4.5 6.8 DS caramel (ammonia-sulfite process) + 639 4.5 TABLE 1. COMPOSITION OF THE TEST DIET (IN g) (con't) Group 1 2 3 4 5 6 7 8 9 10 11 12 Caramel in % 5 10 15 10 10 2 4 6 4 4 0 0 DS caramel (ammonia-sulfite process) + 852 4.5 a SS = Single strength. b Quantity of methylimidazole in ppm. c DS = Double strength. Special studies on teratogenicity Groups of 19-22 pregnant female mice (strain CD-1) were used in this experiment. The mice reeeived by oral intubation 0.16, 74.3, 345 and 1600 mg caramel per kg body weight for 10 consecutive days. The caramel type was described as "beverage" (ammonia-sulfite process). The administration was started on day six continuing daily through day 15 of gestation. Appearance, behaviour, food consumption and growth were studied. On day 17 all dams were subjected to Caesarcan section and the number of implantation sites, resorption sites, liver, dead foetuses and body weight of the pups were recorded. All foetuses were examined grossly for external congenital abnormalities, one-third of the foetuses of each litter underwent visceral examination (Wilson technique), the remaining two-thirds were stained with alizarin for skeletal defects. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls (Morgareidge, 1974a). Virgin adult female rats (strain Wistar) were given by oral intubation 0, 16, 74.3, 345 and 1600 mg caramel/kg body weight for 10 consecutive days. The caramel types was described as "beverage" (ammonia-sulfite process). The administration was started on day six and continuing daily through day 15 of gestation. On day 20 the animals were subjected to Caesarcan section. The same parameters as studied in the mouse experiment were also studied in the rat. No clearly discernible effect on nidation or on maternal or foetal survival was found. The number of abnormalities found in the test groups did not differ from that occurring spontaneously in the sham-treated controls (Morgareidge, 1974a). Virgin adult Dutch-belted female rabbits were given 0, 16, 74.3, 345 and 1600 mg caramel/kg body weight by oral intubation. The caramel type was described as "beverage" (ammonia-sulfite process). The administration started on day six continuing daily through day 18. On day 29 all doses were subjected to Caesarean sections and all the same parameters as mentioned under mouse and rat were studied. No clearly discernible effect on nidation or on maternal or foetal survival were seen. The abnormalities seen in either soft or skeletal tissues of the test groups did not differ from that occurrency spontaneously in the sham-treated controls (Morgareidge, 1974b). A study in mice, rats and rabbits was carried out as described above for mouse, rat and rabbit separately. The caramel was described as "bakers and confectionary caramel". Caramel was given by gavage in doses of 0, 16, 74.3, 345 and 1600 mg/kg body weight during the above stated periods of gestation. No abnormalities were seen (Morgareidge, 1974a and b). Special studies on mutagenicity Caramel (not described what was tested) was evaluated for genetic activity in a series of in vitro microbial assays with and without metabolic activity. Salmonella typhimurium (strain TA-1535, TA-1537 and TA-1538) and Saccharomyces cerevisiae was used. This caramel was not genetically active under the test conditions employed in this study (Brusick, 1974). Special studies on metabolites Male albino mice (20-25 g) were used to determine the median convulsive dose (CD50) and the median lethal dose (LD50) of a few imidazoles. The results are given in Table 4. TABLE 4. CONVULSANT AND LETHAL EFFECTS OF IMIDAZOLES CD50 + SE LD50 + SE in mg/kg bw in mg/kg bw intrap. oral intrap. oral 4-methylimidazole 155 ± 5 360 ± 18 165 ± 3 370 ± 15 1-methylimidazole 380 ± 8.2 1 400 ± 79 380 ± 8.2 1 400 ± 79 2-methylimidazole 500 ± 12 1 300 ± 70 480 ± 18 1 400 ± 114 imidazole 560 ± 34 1 880 ± 45 610 ± 7.4 1 880 ± 45 All the imidazoles tested produced varying degrees of tremor, running, restlessness, siaborrhea, Straub tail, opisthotonus and tonic extensor seizure that ended in death (Nishie et al., 1970). The CD50 and the LD50 of 4-methylimidazole in one-day-old chicks was respectively the intraperitoneal CD50 (± SE) was 174 ± 10 mg and the LD50 210 ± 15 mg/kg body weight. Per orally the CD50 was 580 ± 30 mg and the LD50 was 599 ± 50 mg/kg body weight. Doses of 100 mg/kg intraperitoneally caused tremors, peeping and spreading of the wings. Doses over 150 mg/kg body weight intraperitoneally caused opisthotonus, prostration with clonic leg movements and terminal tonic extensor seizure (Nishie et al., 1970). Acute toxicity LD50 Reference Animal Route mg/kg body weight Rat Oral >2.3 ml equiv. 1 900 Foote et al., 1958 Oral > 25 ml equiv. 17 500 Chacharonis, 1960 Oral >30 ml equiv. 20 400 Chacharonis, 1963 No abnormalities were detected after observation of animals for 14 days following administration of 12 different caramel colour products mostly based on ammonia or ammonium sulfate catalysts (Foote et al., 1958; Chacharonis, 1960; Chacharonis, 1963). A single dose of up to 10 g/kg body weight in mice and 15 g/kg in rabbits of caramels produced by the ammonia catalyser closed pan process or sodium hydroxide process did not cause convulsions or other signs of distress (Sharratt, 1971). Short-term studies Mouse In a four to six week study, albino Swiss mice (10 males and 10 females per group) were given caramel (ammonia process) containing 830 ppm 4-methylimidazole in the following dose levels 0, 1, 2, 4, 8 and 16% in the diet. No influence on appearance, behaviour and food intake were noticed. The growth was especially in the group with 16% decreased in the third and fourth week. The faeces of the animals especially with the higher dose-levels was soft, tarry in appearance and assumed a sticky, pasty consistency and poorly formed. In the males with 16% caramel an increase of neutrophilic leucocytes and a decrease in lymphocytes were noticed. The mean relative weight of the caeca (full weight and empty weight) was partly or clearly increased in the 4, 8 and 16%. About the histopathological study only is mentioned that there were no remarkable findings. Histopathological examinations were not fully reported (Procter, 1976). Rat This study is a part of the experiment described under reproduction studies. It was further the outcome of the interim sacrifice of one-fourth of the animals utilized in the long-term study described below. The rats (10 males and 10 females in each group) were selected from the first litter of parents that were given diets containing different caramels (anmaonia-sulfite process) (three single strength (SS) and three double strength (DS), containing 200-850 ppm of 4-methylimidazole) in different dose levels (5, 10 and 15% (SS) and 2.4 and 6% (DS); furthermore 3 (ammonia-sulfite process) SS-caramels with different methylimida-zole contents were tested in 10% and 3 (ammonia-sulfite process) DS-caramels with different methylimidazole content were tested in 4% in the diet) (see Table 2). Only group 12 was not included in this experiment. The animals were sacrificed after a feeding period of one year. In this study on behaviour, growth, food intake, mortality, liver and kidney function tests, urine composition, organ weights, no adverse effects were found. Haematological indices showed a slight dose-related decrease in total leucocyte counts in females fed SS-earamel containing 202 ppm of methylimidazole. In males no effect was noticed. In a subsequent experiment in which the same SS-caramel was fed to female rats at levels as high as 15 and 30% no indications of decreased leucocyte counts were observed after 4, 8, 12 or 16 weeks. The only finding which is attributed to the feeding of the caramels consisted of increased accumulations of a yellow-brown pigment and pigment laden macrophages in the megenteric lymph nodes of males and females in all caramel groups. Inflammatory or degenerative changes of the lymphoid tissue was not found (Sinkeldam et al., 1975a and b). Three groups of 20 females (Wistar) received the stock diet to which 0, 10 and 20% of ammonia-sulfite process, SS-caramel containing 202 ppm 4-methylimidazole was added. During the second week of the experiment these levels were increased to 15 and 25% and in week seven increased respectively to 25 and 30%. Throughout 16 weeks the diets were administered and followed by a four-week recovery period, without the caramel. The food consumption and growth of the test animals were comparable with the controls. Leucocyte counts, collected after 4, 8, 12 and 16 weeks did not show statistically significant differences among the groups. The relative weights of the caecum, both filled and empty were distinctly increased already after four weeks of caramel feeding. After a recovery period of four weeks the increases had disappeared. The relative weights of the thymus was not affected. Gross examination at autopsy after 16 weeks revealed a dose- related, brown-greenish discoloration of the mesenteric lymph nodes in all test animals of the highest dose level. After the recovery period of four weeks the colour change was less, but still visible. Microscopically the lymph nodes of the test rats showed pigment accumulation which was not noticeably diminished after withdrawal of the caramel for four weeks. These results failed to confirm the decreased leucocyte count which was observed in females fed 5 up to 15% SS-caramel 202 in the one year feeding study (Sinkeldam et al., 1975a). Seven groups of 20 female rats (Wistar) were fed 0, 15 and 30% of three types of caramel containing 1750 of 4-methylimidazole. The caramels were manufactured by using catalysts ammonia. The administration of the caramel was for eight weeks followed by a four- week recovery period. The diets containing caramel caused dose-related decrease in body weights and food efficiency caused diarrhoea. Leucocyte counts after 4, 8, 10 and 12 weeks did not show differences with the controls. The relative weights of the caecum, both filled and empty were considerably increased already after four weeks. After a recovery period of four weeks the increases had disappeared. Gross examination at autopsy after eight weeks revealed a slight, dose-related brown discoloration of the mesenteric lymph nodes, in a few animals of each test group. After recovery periods of two or four weeks the discoloration was less intensive, but still visible. Microscopically the lymph nodes of the test rats showed accumulation of pigment-laden macrophages, which was not noticeably diminished after withdrawal of the caramel for two or four weeks (Sinkeldam and Van der Heyden, 1975/1976). Six groups of 15 rats (CFE-strain) of each sex were given diets for 10 weeks containing 4, 8 or 16% caramel (ammonia-process) (produced by either an "open" or a "closed" pan ammonia process) (no indications are given about the content of 4-methylimidazoles in these caramels). A group of 25 rats of each sex served as controls. There was a decrease in body weight gain at all dietary levels of both caramel types. There were reduced haemoglobin concentrations in the highest dietary level, while in the lower levels this effect was less clear. After 13 weeks in the males of all the dose levels the Hb-content was significantly decreased. In the females only in the 8 and 16% levels. In certain cases, but less consistent, the total number of red blood cells was lowered. The total number of leucocytes was significantly decreased and a lymphocytopenia was present at all levels in association with lower weight of the thymus and the spleen in the 8 and 16% level. The caecum weights were increased compared with the controls. Increased relative liver and kidney weights suggested an effect on these organs. Changes in the weights of other organs were considered to be related to the differences of body weight between the groups. The volumes of urine excreted during a six-hour period without water or in the two-hour period after a water load were lower than the control values. The latter differences were accompanied by higher values for the specific gravity of the urine. The histopathological study did not reveal treatment-related changes (no details are given) (Gaunt et al., 1975). Sixteen groups of rats were given for 10 weeks different dose- levels of three caramel types. Weanling rats (strain Wistar) per group, 15 males and 15 females, except in the control group in which 60 animals of each sex and in the three groups with the highest dose level of caramel, in which 10 animals of each sex were used. The caramels that were tested were single strength ammonium sulfite (MS), double strength ammonium sulfite (DS) and caramel ammonia-process in dose levels of 0.5, 1.0, 2.0, 4.0 and 16.0% in the diet (no data on the presence of 4-methylimidazole are given. Food intake, growth was estimated and haematological examination and histopathological studies were carried out. Especially lymph nodes, thymus, spleen and caecum were studied for distribution of pigment. Another four groups of 10 rats were given basal diet or diet containing 16% of one of the caramels for 10 weeks. At the end of this experiment all the rats received basal diets. Five rats of each sex were killed after seven days and the remainder after 28 days of feeding basal diet (recovery experiment). In the group with 16% DS and 2% ammonia process caramel decreased body weight gain was found. The total leucocyte count showed in the males in the groups with 2, 4 and 16% ammonia process caramel and less clear in 4 and 16% DS caramel was decreased. In the females this was only in the 16% ammonia process caramel. However, the lymphocyte/ neutrophil ratio was significantly decreased in all dose levels ammonia process caramel. The relative liver weight was increased in 2% ammonia and 2% DS caramel and higher levels. Reduced spleen weight was found in 16% ammonia process caramel. Increased relative kidney weight was already seen in the 2% ammonia process caramel, 2% DS and 16% SS-caramel group. Increased caecal weight was only seen in 16% of all the three caramel types. The pigment in the lymph nodes was seen in 16% DS caramel. Microscopically pigments were found in mesenteric, cervical and axillary lymph nodes in the males in the groups with 4 and 16% SS, 4 and 16% DS caramel and only in the mesenteric lymph nodes of the 16% ammonia process caramel. In the females this was only found in the 16% DS caramel and 16% ammonia-process caramel. After the treatment was stopped the white cell counts, cell ratios and the total number of lymphocytes rapidly returned to normal values. This recovery was complete in both sexes by seven days. The increased relative liver weights did not return completely to normal during the recovery phase, the kidneys and spleen weights recovered partly to normal. The caecal weights changed fast to normel in seven days (BIBRA report, 1977). In a 10-week feeding study 17 groups of 15 male and 15 female rats (strain Sprague-Dawley) were given various caramels in different dose levels. The caramels were an ammonia process caramel (containing 830 ppm 4-methylimidazole), single strength ammonia sulfite caramel (SS) (containing 140 ppm 4-methylimidazole) and a double strength ammonia sulfite caramel (DS) (containing 340 ppm 4-methylimidazole). The dose levels that were fed were 1.25, 2.5, 5, 10 and 15% ammonia process caramel and SS or 0.5, 1, 2, 4 and 6% DS caramel. Furthermore two control groups were used. In the animals with ammonia process caramel in levels of 5% and higher the faeces became within two weeks soft, the water content of the faeces was higher. This was also found in the animals with 15% SS caramel. There was no clear profound effect on the increase of body weight, except the 15% ammonia process caramel group, in this group was the clearest effect. From the blood studies it was clear that the ammonia process caramel caused significant reduction in lymphocyte count and a coincident increase in the numbers of segmented neutrophils. This effect was even found in the 1.25% level (but no clear dose-relation). No macroscopic evidence of abnormal pigmentation of the mesenteric lymph nodes were found. Increase in caecal size (empty weight) was consistently evident in animals of both sexes fed ammonia process caramel at the 5, 10 and 15% level. For the groups of animals with caramel SS and DS this was in general not the case. The histopathological study did not reveal changes in the structure of the ileal or caecal mucosae nor in the reticuloendothelial components of the central or peripheral systems. No abnormal pigmentation of the lymph nodes was found (Procter et al., 1976). A 10-week study with 18 groups of 10 male and 10 female rats (strain Wistar) was carried out with three different ammonia caramels viz. ammonium sulfite caramel single strength (SS), ammonium sulfite caramel double strength (DS) and ammonia process caramel. No data on the content of 4-methylimidazole were given. The dose levels were 0 (control), 1.25, 2.5, 5, 10 and 15% SS caramel or ammonia process caramel or 0 (control), 0.5, 1, 2, 4 or 6% DS caramel. All caramels caused loose stools at the highest dose level, most marked with the ammonia process caramel (in the lower dose levels it was mentioned). Body weights were slightly decreased only in the group fed 15% ammonia process caramel, in both sexes. Leucocyte (lymphocyte) counts were decreased in males and females fed 15% ammonia process caramel in all dose levels (in the lower dose levels only in the females). The relative weight of the caecum (both filled and empty) was distinctly increased by feeding ammonia process caramel. With SS and DS caramel only slight indications of caecum enlargement were observed. Minimal amounts of pigment were observed in mesenteric lymph nodes of several rats in the rats with 1.25% of ammonia process caramel 2.5 and 1% of SS and DS caramel respectively and the higher dose levels. From this experiment it appeared that the amonia process caramel was distinctly more active than the two other caramels in regard to growth depression, enlargement of the caecum and decrease in leucocyte counts (Sinkeldam and Van der Heyden, 1976a) Groups of five male and five female rats were given 1 ml/kg body weight of concentrated caramel colour for 21 days. Some diarrhoea was induced in all animals but no other abnormalities were noted. Gross and histopathology revealed no significant changes due to administration of the test compound (Foote et al., 1958). Groups of five rats received either 10 or 20% caramel solution equivalent to about 10 or 20 g/kg body weight as sole source of fluid for 127 days. Only dark faeces and very mild diarrhoea were noted. No adverse effects were noted regarding general health, body weight, food and fluid consumption, haematology, gross and histopathology (Haldi and Wynn, 1951). Six groups of five male and five female weanling rats received 0 or 10% caramel solution as their sole fluid source for 100, 200 or 300 days respectively. No adverse effects were noted regarding growth, food and fluid intake, haematology, gross and histopathology (Haldi and Wynn, 1951). Groups of 16 male and 16 female rats received either 0 or 10% caramel solution for 100 days and groups of five rats received 20% caramel solution for 100 days. At the lower test level there were no observable abnormalities as regards growth, food consumption, haemetology, gross and histopathology. Only growth and haematology were examined at the higher test level (Haldi and Wynn, 1951). Three groups of 20 male and female rats received either 0 or 11-14 g/kg body weight of caramel solutions for 100 days. Growth and food intake did not differ significantly between test and control animals. Gross and histopathology showed no abnormal findings related to administration of the test compound (Haldi, 1958). Four groups of 10 male and 10 female rats received 0, 0.1, 1.0 and 10% of caramel colour in their diet for 12 weeks. No adverse effects were noted on growth, food consumption, urinalysis, haematology, gross and histopathology related to administration of the caramel colour (Prier, 1960). Groups of 10 male and 10 female rats received 0, 5, or 10 g/kg caramel colour in their diet for three months. Weight gain was normal in all groups. Food consumption, haematology and urinalysis were comparable. Gross and histopathology showed no test-related adverse findings (Chacharonis, 1960). Four groups of 10 male and 10 female rats received 0, 5, 10 and 20% of two different caramel colours in their diet for 90 days. In addition, a paired feeding study involving five male rats in two groups was run for 23 days with one sample at the 20% level, and there was no difference in the rate of growth. The only effects attributable to treatment were a mild depression in growth of male rats at the 10 and 20% level due to impalatibility of the test diet. No other adverse findings were noted in growth, behaviour, mortality, haematology, urinalysis, gross pathology, organ weights, and histopathology (Kay and Calandra, 1962). Four groups of 10 male and 10 female rats received either 0 or 10% of three different caramel colours in their diet for 90 days. Weight gains showed slight reduction compared with controls but food consumption was normal for all groups. No abnormalities were noted regarding haematology, urinalysis, gross and histopathology (Chacharonis, 1963). Four groups of 15 male end 15 female rats received 0, 5, 10 and 20% of caramel colour in their diet for 90 days. No adverse effects were noted on appearance, behaviour, survival, body weights, food intake, haematology, blood chemistry, urinalysis, organ weights, gross and histopathology (Oser, 1963). Four groups of 10 male and 10 female rats received 0, 0.015, 0.3 and 3.0% of caramel colour in their diet for 90 days. No differences between test and control animals were noted regarding body weight, food consumption, haematology, urinalysis, gross or histopathology (Nees, 1964). Four groups of rats received 0, 4, 8 and 16% caramel colour in their diet for three months. No convulsions or other behaviour abnormality or signs of neurological damage were seen. No macroscopic or microscopic pathological abnormalities were found in the CNS. Other results are still to come (Sharratt, 1971). Dog Four groups of three male and three female adult beagles received 0, 6, 12.5 and 25% of caramel colour in their diet five days per week for 90 days. No significant adverse effects were noted due to the test compound on growth behaviour, food consumption, mortality, liver function, kidney function, haematology, urine analysis, gross and histopathology (Kay and Calandra, 1962). Long-term studies Rat No formal studies are available but in the course of a reproduction study using 25 pairs of one male and one female rat given 0 or 0.8 g/kg body weight caramel colour in their drinking fluid as part of a beverage tested, several pairs survived for two years or longer. No deleterious effects on growth were noted. The data are rather incomplete (Bachmann et al., 1946). Six different caramels were tested in this long-term toxicity study. Three ammonia-sulfite process single strength (SS) and three ammonia-sulfite process double strength (DS) caramels each containing different quantities of 4-methylimidazole ranging from 200-850 ppm. Each group consisted of 40 male and 40 female rats (Wistar) except the control group, that had the double number of animals. These animals were selected from the first litter of parents, which had been kept on diets containing the various caramels since weaning age (see under reproduction studies). The different dose levels that were tested were 5, 10 and 15% caramel SS and 2, 4 and 6% caramel (DS), furthermore three caramels SS with different methylimidazole contents, tested in 10% and three caramels DS with different methylimidazole contents tested in 470 in the stock diet (see Table 1 on page 48, except that group 12 was not included). After one year, 10 male and female rats in each group were sacrificed. Findings are reported separately (see short-term studies). Observations were made of general appearance, behaviour, mortality, growth and food intake, haematological factors and clinical constituents in blood and urine. After about 14 months considerable mortality was observed both in control and treated groups, probably due to intercurrent disease. Approximately three-quarters of the animals died or were killed before the experiment was terminated at week 104. Organs of dead or killed animals were weighed and extensive histopathological examinations were carried out on all rats of all groups. However, one-third of the animals could not be examined histopathologically due to autolysis. At week 104 organs were weighed and extensive histopathological examinations were carried out on all rats of all groups. In these parameters no abnormalities were found except that the haemoglobin levels and haematocrit values were slightly decreased at week 78 and 98 in males fed the DS caramels. Leucocyte counts were decreased in females fed 15% SS-caramal containing 202 ppm 4-methylimidazole and fed 10% SS-caramel containing 400 ppm 4-methylimidazole at week 13 and 52. At autopsy an increased incidence of greenish discoloured mesenteric lymph nodes was observed in most groups fed high levels of caramel. Again microscopically an increased pigment-phagoeytosis in the mesenteric lymph nodes in all test groups (except the group with 2% caramel DS + 350 ppm 4-methylimidazole) was observed. No evidence of any other adverse structural or cellular alteration was found. Gross and microscopic examination of the other organs did not reveal any pathological changes attributable to the ingestion of the caramels. No increase in the incidence of neoplastic lesions in the different groups was found (Sinkeldam et al., 1976). Four groups of 48 male and 48 female rats (strain Wistar) were given diets containing 0 (control), 1, 3 and 6% of armnonia process caramel (ammonia catalysed "half open - half closed pan" caramel); no indication about the presence of 4-methylimidazole was given) for two years. Besides food and water intake, growth, mortality, haematological examination, urine analysis, kidney function tests, organ weights and histopathological examination were carried out. In both sexes, but significant in the males, there was a decrease in growth at all dose levels. This was accompanied by a reduction in the cumulative food intake. A significant reduction in white cell number (in the 6% group) was associated in the early part of the study with a lymphocytopenia, which was present until week 80 in the male rats fed a diet containing 3 and 6% caramel. In the female animals the lymphocytopenia was present until week 52 in these two groups (the 1% group was not tested). The spleen weight was reduced in a dose-related manner. The relative weight of the (full) caecum was clearly increased at all dose levels. In the histopathologieal findings a few changes are of interest because they may relate to the administration of the caramel. In the pancreas of the control animals no changes were found, while in the test groups (not dose-related) in total 10 hyperplastie changes were found. The number of tumours of the pancreas however, showed no relation to the administration of the caramel. There was no evidence of a carcinogenic effect. The authors concluded that a no-untoward effect could not be established for the caramel used in this particular study (Evans et al., 1976). OBSERVATIONS IN MAN In a number of animal studies a regular finding was an influence on the white blood cells, such as a decrease of the total number of leucocytes and/or a decrease in the number of lymphocytes. A pilot study was carried out in which daily 1.5 g of ammonia process caramel (prepared with ammonia by a closed pan process) was given to nine human volunteers for 21 days. Total circulating leucocytes, lymphocytes and erythrocytes together with haemoglobin concentration were measured prior to and during the treatment. No changes were found that could be attributed to caramel treatment. Another finding in the animal studies is an enlarged caecum and soft stool to diarrhoea. In this human experiment six of the subjects showed no differences from normal in stool frequency or condition. Three volunteers had occasionally soft stools (no control group was used) (BIBRA report 1976). Twenty healthy adult volunteers (10 males and 10 females) were given ammonia sulfite single strength caramel (no data on content of 4-methylimidazole) for three periods of 21 days. The first period they received 6 g/day, the second period 12 g/day and the third period 24 g/day. A full seven-day rest was given between each 21 day period of ingestion. Up till now the results of the first two periods are available and described here. Haematological, clinical biochemical and routine urinary parameters were monitored at the beginning of each ingestion period, after 10 days of ingestion and at the end of each ingestion period. Individual values for haemoglobin, haematocrit, RBC, corrected sedimentation rate, WBC and differentials (neutrophils and band stabs, basophils, eosinophils, monoeytes and lymphoeytes) were found to be within normal limits. For these values there were no changes which may have indicated a trend. There were a few instances of mild neutropenia and mild lymphocytosis but these were not consistent and were unrelated to ingestion of caramel. On the other hand, caramel ingestion was associated with increased frequency of bowel movements and softening or increased liquidity of faeces during the caramel ingestion periods, particularly in the second period when the caramel ingestion level doubled from 6 g/day to 12 g/day (Marier et al., 1977). Twenty healthy adult volunteers (10 males and 10 females) were given anmaonia sulfite double strength caramel (no data on content of 4-methylimidazole) for three periods of 21 days. The first period they received 6 g/day, the second period 12 g/day and the third period 24 g/day. A full seven day rest was given between each 21 day period of ingestion. Up till now the results of the first two periods are available and described here. Haematological, clinical biochemical and routine urinary parameters were studied at the beginning of each ingestion period, after 10 days of ingestion and again at the end of each ingestion period. Most individual values for haemoglobin, haematocrit, RBC, corrected sedimentation rate, WBC and differentials (Neutrophils and band stabs, basophils, eoslnophils, monocytes and lymphocytes) were found to be within normal limits. For these values there were no changes of clinical significance and there were no consistent patterns which may have suggested trends. There were a few instances of values outside the normal range indicating mild neutropenia and mild lymphocytosis but these were observed throughout the two periods including pre-ingestion time, only at pre-ingestion or only as isolated incidents. These changes are not believed to be related to the ingestion of ammonia sulfite caramels. Many subjects experienced frequency of evacuation and softening or increased liquidity of faecal discharges during the two caramel ingestion periods and particularly in the second period when the caramel ingestion level was doubled from 6 g/day to 12 g/day (Marier et al., 1977a). REFERENCES Battelle Memorial Institute (1971) Unpublished report dated 4 May 1971 Bachmann, G. et al. (1946) J. Nutr., 32, 85 Unpublished BIBRA report (1976) A study of the haematological effects of caramel in human volunteers, Report No. 1/172/76 Unpublished BIBRA report (1977) The shortterm (10 week) feeding study with three caramels in rats, Report No. 177/2/77 Brusick, D. (1974) Mutagenic evaluation of compound FDA 71-83, caramel. Unpublished report Litton Bionetics Inc., Dec, LBI project 2468 Chacharonis, P. (1960) Unpublished report No. S.A. 54219 of Scientific Associates Inc. Chacharonis, P. (1963) Unpublished report No. S.A. 79105 of Scientific Associates Inc. Evans, J. G., Butterworth, K. R., Gaunt, I. F. and Grasso, P. (1976) Longterm toxicity study in the rat of a caramel produced by the "half-open - half closed pan" ammonia process. Unpublished BIBRA report No. 6/1976 Foote, W. L., Robinson, R. F. and Davidson, R. S. (1958) Unpublished report of Battelle Memorial Institute Gaunt, I. F.,Lloyd, A. G., Grasso, P., Gangolli, S. D. and Butterworth, K. R. (1975) Toxicological investigations of caramels. I. A shortterm study in the rat with two caramels produced by variations of the "Ammonia process". Unpublished BIBRA report No. 14, 1975 Haldi, J. and 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 Marier, G. and Orr, J. M. (1977) Tolerance study of single strength ammonia sulfite caramel in human volunteers. Unpublished Bio-research Laboratories, Project No. 5612 Marier, G. and Orr, J. M. (1977a) Tolerance study of double strength amonia sulfite caramel in human volunteers. Unpublished Bio-research Laboratories, Project No. 5711 Morgareidge, K. (1974a) Teratologic evaluation of FDA 71-83 (caramel, beverage) in mice, rats and rabbits. Unpublished, Food and Drug Research Lab. Inc. report No. PB 234-867 Morgareidge, K. (1974b) Teratologic evaluation of FDA 71-82 (caramel, bakers and confectioners) in mice, rats and rabbits. Unpublished, Food and Drug Research Lab. Inc. report No. PB 234-870 Nees, P.O. (1964) Unpublished report of Wisconsin Alumni Research Foundation Nishie, K., Waiss, A. C. and Keyl, A. C. (1969) Toxicity of methylimidazoles, Toxicol. Appl. Pharmacol., 14, 301-307 Nishie, K., Waiss, A. C. and Keyl, A. C. (1970) Toxicol. Appl. Pharmacol., 17, 1 Oser, B. L. (1963) Unpublished report No. 83911 of Food and Drug Research Laboratories Prier, R. F. (1960) Unpublished report No. 9070599 of Wisconsin Alumni Research Foundation Procter, B. G. (1976) A preliminary evaluation of the potential toxicological effects of ammonia caramel in mice. Unpublished report of Bio-research Laboratories Ltd., Project No. 5705 Procter, B. G., Berry, G. and Chappel, C. I. (1976) A toxicological evaluation of various caramels fed to albino rats. Unpublished report of Bio-research Laboratories Ltd., Project No. 4244 Sharratt, M. (1971) Unpublished report Sinkeldam, E. J. and Van der Heyden, C. A. (1975a) Shortterm feeding study with Caramel SS 202 in albino rats. Unpublished CIVO report No. R4777 Sinkeldam, E. J. and Van der Heyden, C. A. (1975/1976) Shortterm feeding test with three types of caramels in albino rats. Unpublished CIVO report No. R4789 Sinkeldam, E. J. and Van der Heyden, C. A. (1976a) Shortterm (10 week) feeding study in rats with three different ammonia caramels. Unpublished CIVO report No. R5120 Sinkeldam, E. J., Van der Heyden, C. A. and Beems, R. B. (1976) Chronic (two year) feeding study in rats with six different ammonia caramels. Unpublished CIVO report No. R4961 Sinkeldam, E. J., Willems, M. I. and Van der Heyden, C. A. (1975b) One year feeding study in rats with six different ammonia caramels. Unpublished CIVO report No. R4767 Til, H. P. and Spanjers, M. The (1973) Reproduction study in rats with six different ammonia caramels. Unpublished CIVO report No. R4068
See Also: Toxicological Abbreviations