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PubMed1994

Effects of subchronic exposure to Caramel Colour III on the immune system in mice.

Administration of Caramel Colour III is associated with lymphopenia in laboratory animals, especially if the animals are fed a vitamin B6-deficient diet. Recently, functional immunological alterations in rats exposed to Caramel Colour III have been reported. The component of Caramel Colour III that is responsible for the immunological effects has been shown to be 2-acetyl-4-tetrahydroxybutyl imidazole (THI). In the present study, female Balb/c mice fed a diet with a relatively high vitamin B6 content were exposed to 2 or 10% of a commercial Caramel Colour III preparation with a low THI content (less than 25 ppm) in the drinking water for 9 wk. Although this treatment did not induce a lymphopenia in the exposed mice, flow cytometric analysis of lymphocyte subpopulations demonstrated reductions in the CD4+ and CD8+ cell populations. In addition, the proliferative response of spleen cells to B and T cell mitogens was significantly reduced in the mice exposed to 2% Caramel Colour III. No changes were observed in natural killer cell activity or in the humoral antibody response to a viral antigen. The results indicate that Caramel Colour III that meets the specified limit of less than 25 mg THI/kg may, nevertheless, interfere with the lymphoid system in mice with an adequate vitamin B6 status.

Animals↗
PubMed1988

The effect of pyridoxine on the number of lymphocytes in the blood of rats fed caramel colour (III).

The effect of vitamin E, folic acid, pyridoxine (vitamin B6) and choline on the reduction in circulating lymphocytes in the blood of rats fed Caramel Colour (III) also known as ammonia caramel colour (beer type; AC) has been examined. It was found that the reduction in the number of circulating lymphocytes in rats fed AC could be prevented by the addition of pyridoxine to the diet. The activity of AC in reducing the number of circulating lymphocytes also closely resembled that of the pyridoxine antagonist 4'-deoxypyridoxine. After the isolation and identification of 2-acetyl-4(5)-tetrahydroxybutylimidazole (THI), comparative studies indicated that THI was the component of AC responsible for reducing the number of circulating lymphocytes. Although the effect of AC was reduced or prevented by increasing dietary pyridoxine, the lymphocyte reduction associated with the administration of THI was not materially affected by the dietary level of pyridoxine.

Animals↗
PubMed1989

Effect of oral and parenteral administration of B6 vitamers on the lymphopenia produced by feeding ammonia caramel or 2-acetyl-4(5)-(1,2,3,4-tetrahydroxy)butylimidazole to rats.

The ability of B6 vitamers to prevent the lymphopenic effects of ammonia caramel fed to rats has been evaluated. Diets containing 10 ppm pyridoxine or pyridoxal prevented the lymphopenia produced in rats consuming an 8% (w/v) solution of ammonia caramel, whereas the dietary content of pyridoxamine needed to be increased to 20 ppm to have the same effect. In contrast to the results of the enteral administration of the individual B6 vitamers, pyridoxamine was found to be the most effective vitamer in preventing the ammonia caramel-induced lymphopenia when administered parenterally. However, all the nutritionally active forms of vitamin B6 were able to prevent the depression of the peripheral blood lymphocyte count, which resulted from ingestion of ammonia caramel by rats. The proposal that oral administration of pyridoxine may prevent the intestinal absorption of the lymphopenic constituent of ammonia caramel, 2-acetyl-4(5)-(1,2,3,4-tetrahydroxy)butylimidazole (THI), is discredited, since THI was found to reduce the lymphocyte count after parenteral administration in rats fed 0.04 ppm pyridoxone in the diet and that increased amounts of dietary pyridoxine (10 ppm) could still prevent this effect. These findings further emphasise the important relationship between dietary vitamin B6 content and the lymphopenic effects of ammonia caramel/THI in the rat.

Administration, Oral↗
PubMed1992

Caramel colours--a historical introduction.

Caramel colours used in the manufacture of a wide variety of foods and beverages have been an item of commerce for more than one hundred years. The regulatory history of these additives in the US, the UK, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the EC is reviewed, and an introduction to the safety studies of caramel colours in this issue of Food and Chemical Toxicology is provided.

Candy↗
PubMed1992

Characterization and specifications of caramel colours: an overview.

An overview of the chemical characterization and specifications of the four classes of caramel colour, the historical development of the four classes, and the methods of manufacture is presented. The rationale for the approach used to characterize caramel colour and the derivation of the specifications are given. Results of the characterization programme and the derived specifications are presented in subsequent papers in this issue of Food and Chemical Toxicology.

Candy↗
PubMed1992

Characterization of Caramel Colour IV.

A large number of commercial Caramel Colour IV samples were characterized in order to assess the uniformity of the class and to provide data to be used in specifications development. Owing to the chemical and physical complexity of caramel colour it was not feasible to perform detailed analysis of all constituents for assessment of uniformity. Instead, selected parameters were evaluated and judgements were made with respect to compositional uniformity based on the similarities of these parameters among the various samples. As Caramel Colour IV is required by the food industry in a range of colour intensities, there must be a range of properties that differ from sample to sample, but that are sufficiently similar for the material to still be considered as part of the Caramel Colour IV class. Fractions as well as whole caramel were analysed using selected spectrophotometric, chromatographic and chemical techniques. Samples were fractionated based on molecular weight and polarity. The data presented here provide evidence for the uniformity in composition of Caramel Colour IV with respect to molecular weight distribution, to nitrogen and sulphur content and their distribution throughout the fractions, to absorbance properties and to specific low molecular weight compounds. Thus, it can be concluded that Caramel Colour IV exhibits compositional uniformity within the range of colour intensity required by the food industry worldwide.

Candy↗
PubMed1992

Characterization of Caramel Colours I, II and III.

Groups of representative samples of Caramel Colours I, II, and III were characterized in order to assess the compositional uniformity within each class and to determine differences between each class. Compositional uniformity within each class was demonstrated by fractionation of the caramel samples and analysis by UV and visible absorbance, HPLC and chemical analysis. Although there was overlap between certain parameters of one or more caramel colour classes, the overall 'fingerprint' for each class, based on several different parameters, was distinctly unique. Such parameters included net ionic charge, absorbance properties, nitrogen and sulphur content, specific low molecular weight compounds and HPLC profiles. HPLC analysis revealed unique profiles within each class and distinct differences between classes. The data presented here for Caramel Colours I, II, and III, and elsewhere for Caramel Colour IV, indicate that each of the four classes can be considered as separate and that, throughout each class, the samples characterized constitute a homologous series of mixtures in terms of chemical composition.

Candy↗
PubMed1992

Development of specifications for caramel colours.

Specifications have been developed to define each of the four classes of caramel colour. The specifications were based on analysis of a large database generated during the course of characterization studies of each of the classes. A series of simple and practical tests was developed for the analysis of caramel colour samples to ensure conformity to the specifications.

Candy↗
PubMed1992

Absence of mutagenic activity in Salmonella and of clastogenic activity in CHO cells of Caramel Colours I, II, III and IV.

A total of 15 caramel colours were examined for genotoxic activity using the Salmonella typhimurium plate incorporation assay (Ames test). Five bacterial strains, TA1535, TA1537, TA1538, TA98 and TA100 were used in all the plate incorporation tests. Caramel colours can be divided into four classes, classification depending on the preparative method used. In this study, representatives of all four classes of caramel colour were tested for genotoxic potential in the Ames test, some of the caramel colours being tested both with and without a pre-incubation stage. None of the 15 caramel colours tested exhibited genotoxic potential in any of the five bacterial tester strains. The last two caramel colours tested, in the series of 15 [203-23-4 (Class II) and 311 (Class III)] were also assessed for clastogenic potential. For this test, cultures of CHO cells were exposed to the two caramel colours and metaphase preparations from these cultures examined for evidence of chromosomal aberrations. No evidence of chromosome damaging activity was observed.

Animals↗
PubMed1992

Assessment of the genotoxic potential of Caramel Colour I in four short-term tests.

A battery of three short-term tests in vitro and one in vivo was used to determine the genotoxicity of Caramel Colour I. The results of the bacterial mutation assay, using five strains of Salmonella typhimurium, and the mouse micronucleus assay in vivo showed no evidence of genotoxic activity. Results from both the cytogenetics assay in vitro, using CHO cells, and the mouse lymphoma assay indicated that there was some genotoxic activity associated with Caramel Colour I but only in the absence of S-9 and at very high dose levels.

Animals↗
PubMed1992

Genotoxicity hazard assessment of Caramel Colours III and IV.

Results from a battery of short-term tests in vitro and in vivo used to assess the genotoxicity of caramel colours are presented and discussed in relation to reports from the literature. No evidence of genotoxicity was found in the Salmonella plate incorporation test using five standard strains or in the Saccharomyces cerevisiae gene conversion assay using strain D4, either with or without S-9 for activation. A weak clastogenic effect for a sample of Caramel Colour III in CHO cells was abolished in the presence of S-9. Two samples of Caramel Colour IV were not clastogenic in CHO cells. Salmonella pre-incubation tests without S-9 also failed to reveal any mutagenic activity for any of the caramel colours tested. The Caramel Colour III sample that showed clastogenic activity in CHO cells in vitro did not induce micronuclei when evaluated in a mouse bone marrow assay. These results are in general agreement with reports in the literature regarding the genotoxicity of caramel colours, and support the conclusion that caramel colours do not pose a genotoxic hazard to humans.

Animals↗
PubMed1992

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.

Animals↗
PubMed1992

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)

Administration, Oral↗
PubMed1992

Effects of Caramel Colour III on the number of blood lymphocytes: a human study on Caramel Colour III immunotoxicity and a comparison of the results with data from rat studies.

Administration of the colour additive Ammonia Caramel Colour (Caramel Colour III) to rats has been associated with decreased lymphocyte counts, specifically in rats fed a diet low in vitamin B6. This effect is rapidly reversible and is caused by an imidazole derivative (THI) in Caramel Colour III. In the present paper, the conduct of a human study with Caramel Colour III is outlined and the results of blood lymphocyte counts are presented. No decrease in the number of blood lymphocytes occurred in marginally vitamin B6-deficient humans who consumed Caramel Colour III at the acceptable daily intake level (200 mg/kg body weight/day) for 7 days. These data are discussed in relation to the effects of Caramel Colour III and THI on blood lymphocyte numbers in rats.

Aged↗
PubMed1992

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).

Administration, Oral↗
PubMed1992

Absorption, distribution and excretion of the colour fraction of Caramel Colour IV in the rat.

Caramel Colour IV prepared from [U-14C]glucose was ultrafiltered in order to isolate the high molecular weight colour fraction (HMCF). The colour fraction that was non-permeable to a 10,000-Da porosity membrane, contained 84% of the colour, 22% of the solids and 24% of the radioactivity of the [14C]Caramel Colour IV. The absorption, distribution and excretion of [14C]HMCF were evaluated in male rats after administration of single or multiple oral doses of the material at a dosage level of 2.5 g/kg body weight. Rats on the multiple oral dosage regimen were given unlabelled HMCF in their drinking water for 13 days before the administration of a bolus dose of [14C]HMCF on day 14. On both dosage regimens, the predominant route of excretion was by way of the faeces. Less than 3% of the administered radioactivity was excreted in the urine and only a negligible amount was found in the expired air. More than 99% of the administered radioactivity was excreted within 96 hr. The principal tissues in which radioactivity was found were the mesenteric lymph nodes, liver, kidney and tissues of the gastro-intestinal tract. No major differences were observed in the absorption, distribution or excretion patterns between the single and multiple oral dose regimens.

Absorption↗
PubMed1992

Effects of the colour additive caramel colour III on the immune system: a study with human volunteers.

Administration of the colour additive Caramel Colour III to rats has been associated with decreased numbers of lymphocytes and several other changes in the immune system, as well as in immune function parameters, specifically in animals fed a diet with a relatively low vitamin B6 content. The effects are caused by the imidazole derivative 2-acetyl-4(5)-tetrahydroxybutylimidazole (THI). Caramel Colour III is commonly used in food products such as bakery products, soya-bean sauces, brown sauces, gravies, soup aromas, brown (dehydrated) soups, brown malt caramel blend for various applications, vinegars and beers, and effects in humans on dietary intake cannot be excluded. Elderly male volunteers with a marginal deficit in vitamin B6 were considered a relevant and potentially sensitive group to study possible effects of Caramel Colour III on blood lymphocyte numbers (total and within subsets) or on proliferative responses of lymphocytes to mitogenic stimulation. In addition, several other haematological parameters, as well as serum immunoglobulin levels and immunoglobulin production in vitro by pokeweed mitogen-stimulated mononuclear blood cells were studied. The results of this double-blind intervention study demonstrated that in a selected test group of apparently healthy elderly male volunteers with a biochemically marginally deficient vitamin B6 status, Caramel Colour III containing 23 (commercial sample) or 143 (research sample) ppm THI and administered at the level of the current acceptable daily intake of 200 mg/kg body weight/day for 7 days did not affect any of the factors investigated.

Administration, Oral↗
PubMed1995

Liquorice-induced hypertension--a new understanding of an old disease: case report and brief review.

The case is described of a 40-year-old female with severe hypertension and hypokalaemic metabolic alkalosis, due to prolonged liquorice ingestion. The pseudo-aldosterone-like effects of liquorice have always been attributed to glycyrrhizic acid, but its biochemical substrate has remained elusive. It is now known that glycyrrhetenic acid, the hydrolytic metabolite of glycerrhizic acid, is the active component of liquorice which causes inhibition of the peripheral metabolism of cortisol. Cortisol binds with the same affinity as aldosterone to the mineralocorticoid receptor resulting in a hypermineralocorticoid condition. Ingestion of liquorice may therefore result in retention of sodium and water, hypertension, hypokalaemia, alkalosis and suppression of the renin-aldosterone system. The literature on liquorice-induced hypertension is briefly reviewed with emphasis on the biochemical features of this mineralocorticoid excess syndrome.

Adult↗