Toxicity studies of Caramel Colour III and 2-acetyl-4(5)-tetrahydroxybutylimidazole in F344 rats.

Caramel Colour III is used as a colour additive in beers and a variety of foods. Beer is the most important single source of Caramel Colour III in the diet although consumption of dark beers has been decreasing in recent years. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has established an acceptable daily intake of 200 mg/kg/day for Caramel Colour III. The safety of Caramel Colour III has been questioned during recent years following feeding studies in the rat that were associated with reduced white cell and lymphocyte counts. These effects have been attributed to the presence of 2-acetyl-4(5)-tetrahydroxybutylimidazole (THI) in this class of caramel colour. Short-term oral toxicity studies were conducted on low-THI and high-THI samples of Caramel Colour III (13 wk) and on a sample of THI (28 days). In both studies, the test materials were mixed with demineralized water and the solutions were given to the animals ad lib. in the drinking fluid. In the 13-wk subchronic toxicity study of Caramel Colour III, groups of 20 rats/sex were given concentrations of caramel colour equivalent to intakes of 0, 10, 15 or 20 g low-THI caramel colour/kg body weight/day or 20 g/kg of a high-THI caramel colour. In the 4-wk toxicity study with THI, groups of 20 rats/sex were given 0, 8 or 64 ppm THI (equivalent to approx. 0, 0.9 or 7.2 mg/kg/day) and 10 rats/sex were given 1, 2, 4, 16 or 32 ppm THI (equivalent to approx. 0.1, 0.2, 0.5, 1.9 or 3.7 mg/kg/day) for 4 wk followed by a 2-wk recovery phase for 10 rats/sex in the 0, 8 and 64 ppm groups. Rats given Caramel Colour III had soft faeces; there were no other treatment-related clinical observations and no treatment-related deaths occurred. All treated groups given Caramel Colour III had lower food and fluid consumption than controls. Males given 15 or 20 g low-THI caramel colour/kg or 20 g high-THI caramel colour/kg and females given 20 g/kg of either type had lower body weights than controls. In the 4-wk toxicity study with THI, there were no treatment-related ante-mortem observations, and no effects on body weights or food consumption. Fluid consumption by males and females treated with 64 ppm THI was lower than that of controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Toxicity and carcinogenicity studies of Caramel Colour IV in F344 rats and B6C3F1 mice.

Caramel Colour IV, a type of caramel colour used in the manufacture of cola soft drinks, was evaluated for subchronic and chronic toxicity in rats, and carcinogenicity in Fischer-344 (F344) rats and B6C3F1 mice. In each of the studies, Caramel Colour IV was mixed with demineralized water and the solutions given to the animals ad lib. in the drinking fluid. The concentrations of Caramel Colour IV in the drinking fluid were adjusted periodically to achieve the desired caramel colour intake per kg body weight. In the range-finding studies, groups of 30 rats/sex were given Caramel Colour IV at levels of 0, 15, 20, 25 or 30 g/kg for 13 wk, and groups of 10 male rats were given levels of 0, 2.5, 5, 10 or 15 g/kg for 6 wk followed, for some dose groups, by a 2-wk withdrawal period, and then re-initiation of dosing for another 2 wk. In the rat chronic toxicity study, levels of Caramel Colour IV of 0, 2.5, 5, 7.5 or 10 g/kg were given to groups of 25 rats/sex for 12 months. The test groups in the rat and mouse carcinogenicity studies were composed of 50 animals/sex and each species was given the caramel colour at levels of 0, 0, 2.5, 5 or 10 g/kg for 24 months. In each of the studies, treated animals tended to have dose-related lower water consumption than controls. This was attributed to poor palatability of the drinking fluid, and was generally associated with decreased food consumption and body weights. Rats given caramel colour often had soft or liquid malodorous faeces although there were no treatment-related ante-mortem observations in mice. Blood biochemical changes in the rat (i.e. reduced blood urea nitrogen, alkaline phosphatase and total serum protein) appeared to be related to dietary influences and were not considered toxicologically significant. There were no treatment-related alterations in haematological variables or treatment-related differences in survival or in the incidence of benign or malignant tumours among treated and control groups and no toxicologically important pathological findings. On the basis of these studies, Caramel Colour IV was not toxic or carcinogenic in F344 rats or B6C3F1 mice. The highest dose level tested in the long-term studies (10 g/kg) was considered to be the no-observed-adverse-effect level (NOAEL).

Subchronic toxicity study of Caramel Colour II in F344 rats.

Caramel Colour II is a distinct type of colourant with a pronounced reddish hue. It is made with sulphite reactants but without ammonia. The red colour and a high alcohol solubility provide functional characteristics that are important in foods or beverages containing natural flavour extractives. Caramel Colour II is widely used in ice creams and liqueurs; however, it represents less than 1% of total caramel colour manufacture. The toxicity of Caramel Colour II was evaluated in a 13-wk study in Fischer-344 (F344) rats. The test material was mixed with demineralized water and the solutions were given to the animals ad lib. in the drinking fluid. The concentrations of caramel colour in the drinking fluid were adjusted periodically to achieve the desired caramel colour intake/kg body weight/day. Groups of 20 rats/sex were given Caramel Colour II at levels of 0, 4, 8, 12 or 16 g/kg for at least 13 wk. There were no deaths in any of the groups fed Caramel Colour II. All rats fed caramel colour had soft faeces. All treated groups also had lower fluid consumption that was attributed to poor palatability of the high concentrations of caramel colour that were fed. A number of changes observed (reduced food consumption in all treatment groups except males given 4 g/kg; significantly lower body weights for males given 12 g/kg or more and for females given 8 g/kg or more; lower urine volume and higher specific gravity) were attributed to the reduced water intake and not considered to be toxicologically significant. There were no consistent treatment-related alterations in haematology or blood chemistry variables, and random changes noted were not associated with macroscopic or microscopic pathological alterations. There were no toxicologically important pathological findings. Based on this study, Caramel Colour II was not toxic in F344 rats treated for 13 wk. The highest dose level tested in this study (16 g/kg) was considered to be the no-observed-adverse-effect level.

Subchronic toxicity studies in dogs and in utero rats fed diets containing Bacillus stearothermophilus alpha-amylase from a natural or recombinant DNA host.

Beagle dogs and Fischer 344 rats were fed diets containing 0, 36 or 72 units Bacillus stearothermophilus alpha-amylase (Bsa)/g food or of Bacillus subtilis alpha-amylase (cBsa)/g food. The dogs (four/sex/group) received treated diets for 13 wk. For the rat studies, the parental (F0) generation (12 males and 24 females/group for the Bsa study, and 26 rats/sex/group for the cBsa study) received treated diets for 13 or 4 wk, respectively, before breeding and through weaning of the F1 pups; 20 F1 rats/sex/group received treated diets for at least 13 wk (from weaning until necropsy). There were no treatment-related antemortem observations, reproductive effects or ophthalmic, haematological, macroscopic or microscopic findings in treated dogs or rats, and no differences were noted in body weights for dogs or parental rats. Mean body weights of F1 pups from F0 rats exposed to 72 units cBsa/g were significantly lower than those of the control animals on lactation day 28. This effect may have been related to the slight reduction in body weights and significant reduction in food consumption (gestation days 14-20) of the F0 dams. However, this did not continue into the growth phase for the F1 generation. In the Bsa studies, there were no treatment-related effects in clinical pathology values, and organ-weight data did not correlate with macroscopic or microscopic findings. Male dogs given cBsa had significantly lower albumin (36 units/g), calcium (36 and 72 units/g) and inorganic phosphorus (72 units/g) values compared with those of the control males; there were no treatment-related changes in blood chemistry values in rats. Based on the results of these studies, the no-observable-effect level for alpha-amylase fed to dogs or rats is 36 units/g food.

Subchronic toxicity studies in dogs and in utero-exposed rats fed diets containing Bacillus megaterium amylase derived from a recombinant DNA organism.

Subchronic toxicity studies were performed using a food-grade enzyme product from a recombinant Bacillus subtilis containing the B. megaterium amylase gene. Beagle dogs (four/sex/group) and Fischer 344 rats (25/sex/group) were fed diets containing 0, 20, 60 or 100 units amylase/g food. The dogs received treated diets for 13 wk. The parental (F0) rats received treated diets for 4 wk before breeding and through weaning of the F1 pups; 25 F1 rats/sex/group received treated diets for at least 13 wk (from weaning until necropsy). All animals appeared healthy throughout the studies. Treated animals had sporadic significant differences in body weight and food consumption values when compared with those of controls, but they were not considered toxicologically meaningful. There were no treatment-related effects on reproduction indices, growth variables or litter data in rats. There were no changes in clinical pathology values, organ weights or macroscopic and microscopic observations that were related to treatment. Based on the results of this study, the no-observable-effect level for this amylase fed to dogs or rats is no less than 100 units/g food. This is 6000-12,700 times the predicted human use level.