Oral administration of tartrazine (E102) accelerates the incidence and the development of 7,12-dimethylbenz(a) anthracene (DMBA)-induced breast cancer in rats.

BACKGROUND: Despite the considerable advances made in the treatment of cancer, it remains a global threat. Tartrazine (E102) is a synthetic dye widely used in food industries; it has recently been shown to induce oxidative stress (a well known risk factor of cancer) in rat tissues. The present work therefore aimed to assess the impact of a regular consumption of tartrazine on the incidence of breast cancer in rats. METHODS: Forty (40) Wistar rats aged 55 to 60 days were randomly assigned into 5 groups (n = 8) including two groups serving as normal controls and receiving distilled water (NOR) or tartrazine (NOR + TARZ). The three remaining groups were exposed to the carcinogen DMBA (50 mg/kg) and treated for 20 weeks with either distilled water (DMBA), tartrazine 50 mg/kg (DMBA + TARZ) or a natural dye (DMBA + COL). The parameters evaluated were the incidence, morphology and some biomarkers (CA 15-3, estradiol and α-fetoprotein) of breast cancer. The oxidative status and histomorphology of the tumors were also assessed. RESULTS: A regular intake of tartrazine led to an early incidence of tumors (100% in rats that received TARZ only vs 80% in rats that received DMBA only), with significantly larger tumors (p < 0.001) (mass = 3500 mg/kg and volume = 4 cm3). The invasive breast carcinoma observed on the histological sections of the animals of the DMBA + TARZ group was more developed than those of the DMBA group. The increase in serum α-fetoprotein (p < 0.05) and CA 15-3 (p < 0.01) levels corroborate the changes observed in tumors. The presence of oxidative activity in animals of the DMBA + TARZ group was confirmed by a significant decrease (p < 0.001) in the activity of antioxidant enzymes (SOD and catalase) as well as the level of GSH and increase in the level of MDA compared to the rats of the DMBA and NOR groups. CONCLUSION: Tartrazine therefore appears to be a promoter of DMBA-induced breast tumorigenesis in rats through its oxidative potential. This work encourages further studies on the mechanisms of action of tartrazine (E102) and its limits of use.

Honey attenuates the toxic effects of the low dose of tartrazine in male rats.

Honey is traditionally used in burns, wound healing, ulcers, boils, and fistulas. Honey was tested to prevent tartrazine toxicity in male rats for 8 weeks. The 18 rats of the experiment were randomly divided into three 6-rat groups. The negative control group (G1) fed diet with sulfanilic acid, the tartrazine positive group (G2) fed diet containing tartrazine and sulfanilic acid and the honey-treated group (G3) fed diet as in G2 and cotreated with honey. Tartrazine decreased antioxidants, high-density lipoproteins and proteins, and increased liver enzymes, kidney indices, lipid peroxidation, triglycerides, total cholesterol, and low- and very-low-density lipoproteins. In addition, tartrazine-treated group showed drastic damage of the tissues of stomach, liver, kidney, and testis. Honey treatment increased antioxidants and high-density lipoproteins, and decreased lipid peroxidation, liver enzyme and kidney parameters. Honey treatment also improved stomach, liver, kidney, and testis tissues. In conclusion, honey protects male rats against tartrazine toxicity. PRACTICAL APPLICATIONS: Honey was tested to prevent tartrazine toxicity in male rats in an experiment conducted for 8 weeks. Catalase, glutathione reductase, superoxide dismutase, glutathione reduced, the low- and high-density lipoproteins, lipid peroxidation, liver enzyme, and kidney parameters were measured to evaluate both the toxic effect of tartrazine in G2 and the protective potential of honey in G3.

Tartrazine induced neurobiochemical alterations in rat brain sub-regions.

Tartrazine is a synthetic lemon yellow azo dye primarily used as a food coloring. The present study aimed to screen the neurobiochemical effects of Tartrazine in Wistar rats after administering the Acceptable Daily Intake (ADI) level. Tartrazine (7.5 mg/kg b.w.) was administered to 21 day old weanling rats through oral gavage once daily for 40 consecutive days. On 41st day, the animals were sacrificed and brain sub regions namely, frontal cortex, corpus striatum, hippocampus and cerebellum were used to determine activities of anti-oxidant enzymes viz. Superoxide Dismutase (SOD), Catalase (CAT), Glutathione-Stransferase (GST), Glutathione Reductase (GR) and Glutathione Peroxidase (GPx) and levels of lipid peroxides using Thio-barbituric Acid Reactive Substance (TBARS) assay. Our investigation showed a significant decrease in SOD and CAT activity, whereas there occurred a decline in GST and GR activity with an increase in GPx activity to counteract the oxidative damage caused by significantly increased levels of lipid peroxides. The possible mechanism of this oxidative damage might be attributed to the production of sulphanilc acid as a metabolite in azofission of tartrazine. It may be concluded that the ADI levels of food azo dyes adversely affect and alter biochemical markers of brain tissue and cause oxidative damage.

Developmental Toxicity Assay for Food Additive Tartrazine Using Zebrafish ( Danio rerio) Embryo Cultures.

Tartrazine (TTZ) is an azo dye used as a colorant in food products, drugs, and cosmetics. The present study evaluates the impacts of TTZ on embryonic development of zebrafish ( Danio rerio). Laboratory-raised D. rerio embryos (n = 20/concentration) were exposed to graded dilutions of TTZ (0, 0.1, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 75, and 100 mM) from gastrulation stage (5.25 hours postfertilization [hpf]) until hatching and developmental trajectory was traced up to day 7. The no observed effect concentration (NOEC), median lethal concentration (LC50), median effective concentration (EC50), and teratogenic index (TI) were calculated. Exposure of embryos to < 10 mM TTZ had no effects; 20 to 30 mM TTZ caused tail bending, cardiac and yolk sac edema in 50% of larvae; in 30 to 50 mM TTZ-exposed embryos the heart rates declined along with the above mentioned deformities, causing mortality within 96 to 144 hpf; development ceased completely at 75 to 100 mM concentration. The NOEC and LC50 were recorded at 5 and 29.4 mM dose, respectively. The EC50 values for heart rate, cardiac edema, tail bending, and hatching success were at 59.60, 53.81, 98.28, and 58.97 mM with TI quotient 0.49, 0.54, 0.29, and 0.49, respectively. We conclude that TTZ is not embryo toxic/teratogenic for zebrafish embryos up to a dose level of 10 mM concentration.

Exposure estimate for FD&C colour additives for the US population.

Dietary exposures to the seven food, drug, and cosmetic (FD&C) colour additives that are approved for general use in food in the United States were estimated for the US population (aged 2 years and older), children (aged 2-5 years) and teenage boys (aged 13-18 years) based on analytical levels of the FD&C colour additives in foods. Approximately 600 foods were chosen for analysis, based on a survey of product labels, for the levels of FD&C colour additives. Dietary exposure was estimated using both 2-day food consumption data from the combined 2007-10 National Health and Nutrition Examination Survey (NHANES) and 10-14-day food consumption data from the 2007-10 NPD Group, Inc. National Eating Trends - Nutrient Intake Database (NPD NET-NID). Dietary exposure was estimated at the mean and 90th percentile using three different exposure scenarios: low exposure, average exposure and high exposure, to account for the range in the amount of each FD&C colour additive for a given food. For all populations and all exposure scenarios, the highest cumulative eaters-only exposures in food were determined for FD&C Red No. 40, FD&C Yellow No. 5 and FD&C Yellow No. 6. In addition, the eaters-only exposure was estimated for individual food categories in order to determine which food categories contributed the most to the exposure for each FD&C colour additive. Breakfast Cereal, Juice Drinks, Soft Drinks, and Frozen Dairy Desserts/Sherbet (also referred to as Ice Cream, Frozen Yogurt, Sherbet (including Bars, Sticks, Sandwiches)) were the major contributing food categories to exposure for multiple FD&C colour additives for all three populations.

All India survey for analyses of colors in sweets and savories: exposure risk in Indian population.

In the present study, an attempt has been made to understand the exposure assessment of food colors through 2 major groups, sweets and savories, at a national level so as to evolve a scientific yardstick to fix levels of colors in commodities based on technological and safety requirement. A vast majority of colored food commodities (83.6%) were found to employ permitted colors and confirmed a marked decline in the trend of use of nonpermitted colors (NPCs). Of the 4 zones of India, East zone showed the maximum adulteration (80.3%) both by exceeding the prescribed limits of permitted colors (72.3%) and the use of NPCs (28.7%). Tartrazine was the most popular color among the permitted list, which ranged from 12.5 to 1091 mg/kg. Rhodamine B was the most prevalent dye in the NPCs group. On the basis of average consumption of food commodities and average levels of detected colors, the intake of Sunset Yellow FCF saturates the acceptable daily intake limit to a maximum of 47.8% in children, which is a cause of concern. The uniform maximum permissible limit of synthetic colors at 100 mg/kg under the Indian rules thus needs to be reviewed and should rather be governed by the technological necessity and the consumption profiles of food commodities so that the vulnerable population should not unnecessary be exposed to excessive amounts of synthetic colors to pose health risks.

Effect of food azo dye tartrazine on learning and memory functions in mice and rats, and the possible mechanisms involved.

UNLABELLED: Tartrazine is an artificial azo dye commonly used in human food and pharmaceutical products. The present study was conducted to evaluate the toxic effect of tartrazine on the learning and memory functions in mice and rats. Animals were administered different doses of tartrazine for a period of 30 d and were evaluated by open-field test, step-through test, and Morris water maze test, respectively. Furthermore, the biomarkers of the oxidative stress and pathohistology were also measured to explore the possible mechanisms involved. The results indicated that tartrazine extract significantly enhanced active behavioral response to the open field, increased the escape latency in Morris water maze test and decreased the retention latency in step-through tests. The decline in the activities of catalase, glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) as well as a rise in the level of malonaldehyde (MDA) were observed in the brain of tartrazine-treated rats, and these changes were associated with the brain from oxidative damage. The dose levels of tartrazine in the present study produced a few adverse effects in learning and memory functions in animals. The mechanisms might be attributed to promoting lipid peroxidation products and reactive oxygen species, inhibiting endogenous antioxidant defense enzymes and the brain tissue damage. PRACTICAL APPLICATION: Tartrazine is an artificial azo dye commonly used in human food and pharmaceutical products. Since the last assessment carried out by the Joint FAO/WHO Expert Committee on Food Additives in 1964, many new studies have been conducted. However, there is a little information about the effects on learning and memory performance. The present study was conducted to evaluate the toxic effect of tartrazine on the learning and memory functions in animals and its possible mechanism involved. Based on our results, we believe that more extensive assessment of food additives in current use is warranted.

Safety of ingestion of yellow tartrazine by double-blind placebo controlled challenge in 26 atopic adults.

BACKGROUND: Yellow dye tartrazine is a potential cause of exacerbations of asthma, allergic rhinitis and urticaria in atopic patients. The Brazilian Sanitary Surveillance Agency (ANVISA) published a consultation about the possibility of issuing a label warning addressing these potential effects of food and drugs containing tartrazine. The present study aims to evaluate tartrazine dye safety in atopic subjects suffering from allergic rhinitis, asthma, urticaria or sensitivity to non-steroidal anti-inflammatory drugs (NSAIDs). METHODS: Atopic patients with allergic rhinitis, asthma, urticaria or pseudo-allergic reactions to non-steroidal anti-inflammatory drugs were studied (n=26). The gold standard, double-blind placebo controlled, crossed-over challenge was used RESULTS: There were no statistical differences between placebo and drug in cutaneous, respiratory or cardiovascular aspects. CONCLUSIONS: In a group of atopic subjects with allergic rhinitis, asthma, urticaria or pseudo-allergic reactions to non-steroidal anti-inflammatory drugs, the administration of 35 mg of the tartrazine dye did not precipitate any kind of significant cutaneous, respiratory or cardiovascular reactions when compared to placebo.

Pattern of intake of food additives associated with hyperactivity in Irish children and teenagers.

A double-blind randomized intervention study has previously shown that a significant relationship exists between the consumption of various mixes of seven target additives by children and the onset of hyperactive behaviour. The present study set out to ascertain the pattern of intake of two mixes (A and B) of these seven target additives in Irish children and teenagers using the Irish national food consumption databases for children (n = 594) and teenagers (n = 441) and the National Food Ingredient Database. The majority of additive-containing foods consumed by both the children and teenagers contained one of the target additives. No food consumed by either the children or teenagers contained all seven of the target food additives. For each additive intake, estimates for every individual were made assuming that the additive was present at the maximum legal permitted level in those foods identified as containing it. For both groups, mean intakes of the food additives among consumers only were far below the doses used in the previous study on hyperactivity. Intakes at the 97.5th percentile of all food colours fell below the doses used in Mix B, while intakes for four of the six food colours were also below the doses used in Mix A. However, in the case of the preservative sodium benzoate, it exceeded the previously used dose in both children and teenagers. No child or teenager achieved the overall intakes used in the study linking food additives with hyperactivity.

Estimates of dietary exposure of children to artificial food colours in Kuwait.

To assess the intake of artificial food colour additives by 5-14-year-old children in the State of Kuwait, a 24-h dietary recall was conducted twice on 3141 male and female Kuwaiti and non-Kuwaiti children from 58 schools. The determination of colour additives in 344 foods items consumed was performed using high-performance liquid chromatography with diode array detector. A comparison with the Food and Agriculture Organization and World Health Organization acceptable daily intakes (ADIs) was undertaken to evaluate the potential risk associated with the consumption of artificial colour additives by children in Kuwait. The results indicated that out of nine permitted colours, four exceeded their ADIs by factors of 2-8: tartrazine, sunset yellow, carmoisine and allura red. Further, follow-up studies to provide insight into potential adverse health effects associated with the high intakes of these artificial colour additives on the test population are warranted.

Exposure assessment to synthetic food colours of a selected population in Hyderabad, India.

An exposure assessment of synthetic food colours was carried out among 1-5- and 6-18-year-old individuals by the food frequency method. Children had an intake of solid food consumption in the range 2-465 g day(-1) and liquid food consumption in the range 25-840 ml day(-1) with added colours. Among the eight permitted colours in India, six were consumed by the subjects of the study. The intakes of some subjects exceeded the acceptable daily intake for colours such as tartrazine, sunset yellow and erythrosine, which is 7.5, 2.5 and 0.1 mg kg(-1) body weight, respectively. Therefore, a uniform permissible limit of 100 mg kg(-1) prescribed under the Prevention of Food Adulteration Act in India for all foods is not justified. The limits need to be revised according to the Codex Alimentarius Commission, which permits different maximum levels of additives to various food categories based on both the extent of consumption and the technological justification for its use.

Urinary metabolites of histamine and leukotrienes before and after placebo-controlled challenge with ASA and food additives in chronic urticaria patients.

BACKGROUND: The recovery of mediator metabolites from urine has the potential to provide a rapid, safe, and easily available index of release of mediators. We aimed to determine urinary metabolites of both histamine and leukotrienes (LTs) in patients affected by chronic urticaria (CU). METHODS: Twenty patients with CU were studied. They were selected on the basis of double-blind placebo-controlled challenge (DBPC) with acetyl salicylic acid (ASA) and food additives. Ten patients (group B) were negative to both challenges. Ten patients (group C) presented urticaria and/or the appearance of angioedema during or 24 h after challenge, with reactions to ASA (five patients) or food additives (five patients). We recruited 15 healthy volunteers as controls (group A). During a second challenge, groups B and C were challenged double-blind with a single dose of ASA, or a specific food additive, or placebo. The healthy group was challenged only with a placebo (talc capsule). Patients in groups B and C were challenged twice: with placebo (as groups B1 and C1) and with ASA (groups B2 and C2) or food additives (C2). Four samples of urine were collected; one during the night before the specific or sham challenge (baseline), and three at 2, 6 and 24 h after the challenge. Urinary methylhistamine (N-MH) and LTE4 were analyzed and normalized for urinary creatinine. RESULTS: For urinary N-MH at baseline, there was a significant difference only between group A and groups B1, B2, C1 and C2 (A vs. B1, P < 0.0001; A vs. B2, P < 0.0001; A vs. C1, P < 0.0001; A vs. C2, P < 0.0001). We detected a significant variation in urinary methylhistamine excretion only in group C2 after 2 h, 6 h and 24 h (P < 0.0001). However, no variations were observed in N-MH excretion rate in the other groups (A, B1, C1) after challenge with placebo, and in B2 after challenge with ASA 20 mg. For urinary LTE4 at baseline no differences were found between the mean values for the different groups. After specific challenge, only C2 patients showed significantly increased excretion rates of urinary LTE4 compared with the other groups challenged with placebo (A, B1, C1), or ASA (B2) (P < 0.0001). No significant correlation was seen between urinary LTE4 and methylhistamine excretion rate in any patients. CONCLUSION: Our results show that urinary excretion of N-MH and LTE4 is different for CU patients without ASA or food hypersensitivity, compared to those with CU with ASA or food additive hypersensitivity after specific challenge.

Tartrazine exclusion for allergic asthma.

BACKGROUND: Tartrazine is the best known and one of the most commonly used food additives. Food colorants are also used in many medications as well as foods. There has been conflicting evidence as to whether tartrazine causes exacerbations of asthma with some studies finding a positive association especially in individuals with cross-sensitivity to aspirin. OBJECTIVES: To assess the overall effect of tartrazine (exclusion or challenge) in the management of asthma. SEARCH STRATEGY: A search was carried out using the Cochrane Airways Group specialised register. Bibliographies of each RCT was searched for additional papers. Authors of identified RCTs were contacted for further information for their trials and details of other studies. SELECTION CRITERIA: RCTs of oral administration of tartrazine (as a challenge) versus placebo or dietary avoidance of tartrazine versus normal diet were considered. Studies which focused upon allergic asthma, were also included. Studies of tartrazine exclusion for other allergic conditions such as hay fever, allergic rhinitis and eczema were only considered if the results for subjects with asthma were separately identified. Trials could be in either adults or children with asthma or allergic asthma (e.g. sensitivity to aspirin or food items known to contain tartrazine). DATA COLLECTION AND ANALYSIS: Study quality was assessed and data abstracted by two reviewers independently. Outcomes were analysed using RevMan 4.1.1. MAIN RESULTS: Ninety abstracts were found, of which 18 were potentially relevant. Six met the inclusion criteria, but only three presented results in a format that permitted analysis and none could be combined in a meta-analysis. In none of the studies did tartrazine challenge or avoidance in diet significantly alter asthma outcomes. REVIEWER'S CONCLUSIONS: Due to the paucity of available evidence, it is not possible to provide firm conclusions as to the effects of tartrazine on asthma control. However, the six RCTs that could be included in this review all arrived at the same conclusion. Routine tartrazine exclusion may not benefit most patients, except those very few individuals with proven sensitivity.

[Effect of an oligo-antigen diet on the behavior of hyperkinetic children]. / Der Einfluss einer oligoantigenen Diät auf das Verhalten von hyperkinetischen Kindern.

The influence of an oligoantigenic diet on different dimensions of the behavior of 21 children diagnosed as having attention-deficit hyperactivity disorder (ADHD) was examined. Treatment effects were assessed with three subjective measures (two questionnaires and an interview) and three objective measures (two attention tests and actometer). The study was divided into three phases: baseline, diet and provocation, each lasting three weeks. A crossover design was used. A significant effect was found for the subjective measures, but not for the objective measures. The results are discussed in terms of possible types of effects, e. g. rater effects and environmental effects. It may be that the oligoantigenic diet influences only certain dimensions of hyperactivity.

Allergy to tartrazine in alprazolam.

Allergy to tartrazine-containing psychotropic medication (especially antidepressants) had been reported. 20 patients of apparent allergy to tartrazine-containing alprazolam brands in 480 patients exposed to the dye are described. Rechallenge with non tartrazine-containing alprazolam brands did not produce the similar allergic reactions.

Synthetic food coloring and behavior: a dose response effect in a double-blind, placebo-controlled, repeated-measures study.

OBJECTIVE: To establish whether there is an association between the ingestion of synthetic food colorings and behavioral change in children referred for assessment of "hyperactivity." PARTICIPANTS: From approximately 800 children referred to the Royal Children's Hospital (Melbourne) for assessment of suspected hyperactivity, 200 were included in a 6-week open trial of a diet free of synthetic food coloring. The parents of 150 children reported behavioral improvement with the diet, and deterioration on the introduction of foods noted to contain synthetic coloring. A 30-item behavioral rating inventory was devised from an examination of the clinical histories of 50 suspected reactors. Thirty-four other children (23 suspected reactors, 11 uncertain reactors) and 20 control subjects, aged 2 to 14 years, were studied. DESIGN: A 21-day, double-blind, placebo-controlled, repeated-measures study used each child as his or her own control. Placebo, or one of six dose levels of tartrazine (1, 2, 5, 10, 20, 50 mg), was administered randomly each morning, and behavioral ratings were recorded by parents at the end of each 24 hours. RESULTS: The study identified 24 children as clear reactors (19 of 23 "suspected reactors," 3 of 11 "uncertain reactors," and 2 of 20 "control subjects"). They were irritable and restless and had sleep disturbance. Significant reactions were observed at all six dose levels. A dose response effect was obtained. With a dose increase greater than 10 mg, the duration of effect was prolonged. CONCLUSION: Behavioral changes in irritability, restlessness, and sleep disturbance are associated with the ingestion of tartrazine in some children. A dose response effect was observed.

A method to control particle size of cellulose acetate trimellitate microspheres.

A method for controlling microsphere particle size by regulating the ratio of polymer to solvent concentration and volume fraction, for an emulsion-solvent removal type of microencapsulation system, has been investigated. Viscosity of the external phase was kept constant by using light mineral oil in all experiments. Viscosity of the polymer solution, the internal phase, was modified by changing the ratio of the polymer to solvent concentration. Microspheres were obtained by adding the internal phase to the external phase and stirring the mixture for 30 min. A non-solvent was then added to the system to harden the polymer and recover the microspheres. Polymer concentration was modified by adding extra solvent to the mineral oil, just before the addition of the internal phase and also by adding extra solvent to the polymer phase. Similar, but not identical, results were obtained in both of these systems. A plot of particle size versus polymer to the solvent ratio resulted in sigmoidal curves. The term solvent means the solvent available to the polymer after mixing with mineral oil. A separate curve was obtained for each polymer concentration used in the experiments. When the internal phase volume fraction was incorporated as a variable in the plot of particle size, the three sigmoidal curves merged into a single curve, irrespective of the polymer concentration. The equation developed for controlling the particle size, as a function of polymer and solvent concentration and phase volume ratio, also was tested in systems that contained tartrazine as a model drug. Dissolution experiments were carried out and dissolution rate was correlated to particle size. Microspheres size was controlled by polymer and solvent concentration and phase volume ratio.

Study of the teratogenic potential of FD & C yellow No. 5 when given in drinking-water.

FD & C Yellow No. 5 was available to pregnant Osborne-Mendel rats throughout gestation at dose levels of 0.05, 0.1, 0.2, 0.4 or 0.7% in solution in distilled drinking-water. Based on fluid consumption, the rats received 67.4, 131.8, 292.4, 567.9 and 1064.3 mg FD & C Yellow No. 5/kg body weight/day. Distilled water served as the control. No dose-related changes were seen in mean daily food consumption or maternal body-weight gain. Starting during the second trimester of gestation, fluid consumption was significantly greater in the rats given 0.7% FD & C Yellow No. 5 than in the controls. The females were killed on gestation day 20. No dose-related changes were seen in maternal clinical findings, implantations, foetal viability or foetal size (weight and length). No dose-related foetal terata were seen. Neither visceral development nor skeletal development (sternebral and other skeletal bones) was affected by the dye. The small numbers of statistically significant increases in skeletal variations in the 0.05 and 0.4% levels are considered random because they are not dose related.

Study of the teratogenic potential of FD & C Yellow No. 5 when given by gavage to rats.

FD & C Yellow No. 5 (tartrazine) was given to Osborne-Mendel rats by gavage at dose levels of 0, 60, 100, 200, 400, 600 or 1000 mg/kg body weight/day on days 0-19 of gestation. No maternal or developmental toxicity was observed when the rats were killed on day 20. The mean daily food consumption for the entire period of gestation was significantly greater in the females given 1000 mg/kg body weight/day than in the controls, but maternal body-weight gain was not affected. No dose-related effects were observed in implantations, foetal viability or external foetal development. Foetal skeletal and visceral development was similar among foetuses from all groups. At the doses given, FD & C Yellow No. 5 was neither toxic nor teratogenic.

A double-blind assessment of additive intolerance in children using a 12 day challenge period at home.

Alleged food-additive intolerance (respiratory, dermatological, behavioural or abdominal) was assessed in 19 children using daily challenge drinks of either, base product alone, base product plus sunset yellow/tartrazine, or base product plus sodium metabisulphite/sodium benzoate. The same type of drink was given for 12 days, double-blind and in random order. During the trial the children were maintained on an additive-free diet under supervision. Diary cards were used to record symptoms and medication usage. If there was an apparent association between symptoms and drink ingredient the trial was repeated, again double-blind. Additive intolerance was confirmed by a consistent deterioration of symptoms in only three children. In one, urticaria was induced by the colourings, in another extremely abnormal behaviour was induced by the preservatives and a third child was only free of asthma and abdominal pain on placebo. This form of individual trial, using 12 daily drinks, overcomes some of the objections to a single challenge study. Despite this, intolerance to the additives was only confirmed in 3/19 children in whom it had been believed to occur.