Integration of evidence to evaluate the potential for neurobehavioral effects following exposure to USFDA-approved food colors.

California's Office of Environmental Health Hazard Assessment was tasked with conducting risk assessments for United States Food and Drug Administration-approved food dyes relative to neurobehavioral concerns. The purpose of this assessment was to evaluate the evidence for neurodevelopment effects based on three streams of evidence: 1) studies identified by OEHHA for consideration in a quantitative risk assessment; 2) studies relevant to understanding mechanisms of neurobehavioral effects; 3) an in silico assessment of the bioavailability of USFDA-approved food dyes. The results indicate a lack of adequate or consistent evidence of neurological effects, supported by a lack of bioavailability and brain penetration predicted by the in silico assessment. Further, the mechanistic evidence supports a lack of activity from in vitro neurotransmitter assays, and a lack of evidence to support molecular initiating events or key events in adverse outcome pathways associated with neurodevelopmental effects, supporting a lack of biological plausibility for neurobehavioral effects following food exposures to colors. These conclusions are consistent with other authoritative bodies, such as JECFA and EFSA, that have determined (i) other effects are more appropriate for estimating acceptable daily intakes and (ii) evidence from the neurobehavioral studies lack the strength to be relied upon for quantitative risk assessment.

Development and Validation of HPLC Method for Determination of Sodium Copper Chlorophyllin - A Food Colorant and Its Application in Pharmacokinetic Study.

A simple, accurate, and precise bioanalytical method was developed and validated for the determination of pharmacokinetic parameters of sodium copper chlorophyllin, a USFDA approved food additive and colorant in rat plasma. The column used was Luna® C18 250×4.6 mm, 100 Å, having particle size 4.5 µm, and the mobile phase used was methanol (MeOH), and 10 mM ammonium acetate buffer in the ratio of 90 : 10, the flow rate was 1 ml/min, and the injection volume of 20 µL. The retention time of sodium copper chlorophyllin was obtained at 9 min. The method was found to be linear at the range of 0.50-8.00 µg mL-1 .

HPLC-hrTOF-MS study of copper chlorophylls: Composition of food colorants and biochemistry after ingestion.

Despite the daily consumption of copper chlorophylls (E-141i), the green food colorants in foods high in fats, there is a general need for knowledge regarding their exact composition. Consequently, we have analyzed by HPLC-ESI(+)/APCI(+)-hrTOF-MS2 the accurate composition of different commercial copper chlorophyll colorants for the first time. Data showed a favored yield of copper pheophytins from a series, while pheophytins from b series are preferentially no complexed with copper. The copper pheophytins present in the food colorants consisted mainly of three structural rearrangements. New fragmentation patterns and structural assignments have been described for several copper pheophytins. During the ingestion of copper chlorophylls, no chlorophyll derivative was present in serum nor urine except a new copper-pyroporphyrin a accumulated in a few livers. In any case, this green additive could represent the ideal food colorant, as most of the copper pheophytins are excreted in the feces showing almost no absorption of copper-chlorophylls compounds.

Quantitative Measure of Intestinal Permeability Using Blue Food Coloring.

BACKGROUND: Loss of intestinal barrier integrity plays a fundamental role in the pathogenesis of various gastrointestinal diseases and is implicated in the onset of sepsis and multiple organ failure. An array of methods to assess different aspects of intestinal barrier function suffers from lack of sensitivity, prolonged periods of specimen collection, or high expense. We have developed a technique to measure the concentration of the food dye FD&C Blue #1 from blood and sought to assess its utility in measuring intestinal barrier function in humans. MATERIALS AND METHODS: Four healthy volunteers and 10 critically ill subjects in the intensive care unit were recruited in accordance with an institutional review board approved protocol. Subjects were given 0.5 mg/kg Blue #1 enterally as an aqueous solution of diluted food coloring. Five blood specimens were drawn per subject: 0 h (before dose), 1, 2, 4, and 8 h. After plasma isolation, organic extracts were analyzed by high-performance liquid chromatography/mass spectrometry detecting the presence of unmodified dye. RESULTS: We found no baseline detectable absorption in healthy volunteers. After including the subjects in the intensive care unit, we compared dye absorption in the six subjects who met criteria for septic shock with the eight who did not. Septic patients demonstrated significantly greater absorption of Blue #1 after 2 h. CONCLUSIONS: We have developed a novel, easy-to-use method to measure intestinal barrier integrity using a food grade dye detectable by mass spectrometry analysis of patient blood following oral administration.

Water-in-oil-in-water emulsion obtained by glass microfluidic device for protection and heat-triggered release of natural pigments.

Anthocyanins and norbixin are natural pigments used in food; however, they are unstable. The aim of this study was to evaluate the microencapsulation technique to protect these pigments. Elderberry extract (source of anthocyanins) and norbixin were encapsulated using a microfluidic device with palm oil as middle phase in a water-in-oil-in-water emulsion. The formulations were characterized for morphology, particle size, encapsulation efficiency, zeta potential, color release under heating, Fourier transform infrared spectrophotometry, and color stability under different conditions. Spherical, mononucleated microcapsules, with particle size of 187-190 µm (elderberry) and 164-184 µm (norbixin), and with encapsulation efficiencies values of 47.80-54.87% (elderberry) and 49.18-74.73% (norbixin) were obtained. The formulations showed high color retention, with the encapsulated elderberry extract stored at pH 3.0 being the most stable. This study shows that the microencapsulation of these pigments using a microfluidic device provided protection, and represents a new method for anthocyanins and norbixin delivery in foods.

Pharmacokinetics of Rhodamine 110 and Its Organ Distribution in Rats.

Rhodamine dyes have been banned as food additives due to their potential tumorigenicity. Rhodamine 110 is illegal as a food additive, although its pharmacokinetics have not been characterized, and no accurate bioanalytical methods are available to quantify rhodamine 110. The aim of this study was to develop and validate a fast, stable, and sensitive method to quantify rhodamine 110 using high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) to assess its pharmacokinetics and organ distribution in awake rats. Rhodamine 110 exhibited linear pharmacokinetics and slow elimination after oral administration. Furthermore, its oral bioavailability was approximately 34-35%. The distribution in the liver and kidney suggests that these organs are primarily responsible for rhodamine 110 metabolism and elimination. Our investigation describes the pharmacokinetics and a quantification method for rhodamine 110, improving our understanding of the food safety of rhodamine dyes.

Pharmacokinetics and Biodistribution of the Illegal Food Colorant Rhodamine B in Rats.

The International Agency for Research on Cancer (IARC) demonstrated rhodamine B as a potential carcinogen in 1978. Nevertheless, rhodamine B has been illegally used as a colorant in food in many countries. Few pharmacokinetic and toxicological investigations have been performed since the first pharmacokinetic study on rhodamine B in 1961. The aims of this study were to develop a simple and sensitive high-performance liquid chromatography method with fluorescence detection for the quantitative detection of rhodamine B in the plasma and organs of rats and to estimate its pharmacokinetics and biodistribution. The results demonstrated that the oral bioavailabilities of rhodamine B were 28.3 and 9.8% for the low-dose and high-dose exposures, respectively. Furthermore, rhodamine B was highly accumulated in the liver and, to a lesser extent, the kidney, but was undetectable in the brain. These results provide useful information for improving the pharmacokinetics and biodistribution of rhodamine B, supporting additional food safety evaluations.

Preparation of the chitosan grafted poly (quaternary ammonium)/Fe3O4 nanoparticles and its adsorption performance for food yellow 3.

Chitosan and its derivatives can be used to modify magnetic materials to promote the adsorption properties of the magnetic materials and avoid the weakness of chitosan and its derivatives. In the present study, chitosan grafted poly(trimethyl allyl ammonium chloride) (CTS-g-PTMAAC) was prepared by graft copolymerization; then it was coated on the surfaces of the sodium citrate coated Fe3O4 nanoparticles (SC-Fe3O4) to prepare a novel composite CTS-g-PTMAAC/SC-Fe3O4 magnetic nanoparticles, with which possesses abundant surface positive charges. The structure and properties of the CTS-g-PTMAAC/SC-Fe3O4 composite magnetic nanoparticles were characterized by FTIR, TEM, VSM, and zeta potential. The dye adsorption characteristics of the CTS-g-PTMAAC/SC-Fe3O4 nanoparticles were determined using the food yellow 3 aqueous solutions as a model food effluent. Effect of pH of the dye solution on the adsorption of food yellow 3 was determined and compared with N-2-hydroxylpropyl trimethyl ammonium chloride chitosan coated sodium citrate-Fe3O4 (CTS-g-HTCC/SC-Fe3O4) composite magnetic nanoparticles. The adsorption kinetics, adsorption isotherms, adsorption thermodynamics, and desorption and reusability of the magnetic nanoparticles were investigated.

Health safety issues of synthetic food colorants.

Increasing attention has been recently paid to the toxicity of additives used in food. The European Parliament and the Council published the REGULATION (EC) No. 1333/2008 on food additives establishing that the toxicity of food additives evaluated before 20th January 2009 must be re-evaluated by European Food Safety Authority (EFSA). The aim of this review is to survey current knowledge specifically on the toxicity issues of synthetic food colorants using official reports published by the EFSA and other available studies published since the respective report. Synthetic colorants described are Tartrazine, Quinoline Yellow, Sunset Yellow, Azorubine, Ponceau 4R, Erythrosine, Allura Red, Patent Blue, Indigo Carmine, Brilliant Blue FCF, Green S, Brilliant Black and Brown HT. Moreover, a summary of evidence on possible detrimental effects of colorant mixes on children's behaviour is provided and future research directions are outlined.

The synthetic food colouring agent Allura Red AC (E129) is not genotoxic in a flow cytometry-based micronucleus assay in vivo.

The safety of several azo colouring agents, used as food additives, has during the years been questioned. Allura Red AC (E129) has in some publications been classified as genotoxic. In fact, in the European Union, Allura Red is permitted as a food additive in human food, but, surprisingly, it was not acceptable as an additive for use in animal feed. In this study we have evaluated whether Allura Red is genotoxic using a flow cytometer-based micronucleus assay in peripheral blood of mice. Male FVB mice were given a single intra-peritoneal injection of various doses of Allura Red and sacrificed at 46 h after treatment. The tested doses were 0, 100, 200, 400, 600, 800, 1000, 1500, and 2000 mg/kg body weight (b.w.). Each dose group constituted three mice, except for in the dose group of 1000 mg/kg b. w., which constituted four mice. Blood samples were collected and the frequency of micronucleated polychromatic erythrocytes (fMNPCE) and the cell proliferation (%PCE) was determined. The analyses did not show any significant difference in the %PCE or in the fMNPCE. Consequently, under the testing circumstances one can conclude that Allura Red is not genotoxic.

Mechanisms of behavioral, atopic, and other reactions to artificial food colors in children.

This review examines the research on mechanisms by which artificial food colors (AFCs) and common foods may cause behavioral changes in children with and without attention-deficit/hyperactivity disorder (ADHD). Children with ADHD show excess inattention, impulsivity, and hyperactivity. Studies have shown that a subgroup of children (with or without ADHD) react adversely to challenges with AFCs. Many early studies found few children who reacted to challenges with 20-40 mg of AFCs. However, studies using at least 50 mg of AFCs showed a greater percentage of children who reacted to the challenge. Three types of potential mechanisms are explored: toxicological, antinutritional, and hypersensitivity. Suggestions for future studies in animals and/or children include dose studies as well as studies to determine the effects of AFCs on the immune system, the intestinal mucosa, and nutrient absorption. Given the potential negative behavioral effects of AFCs, it is important to determine why some children may be more sensitive to AFCs than others and to identify the tolerable upper limits of exposure for children in general and for children at high risk.

In vivo tissue uptake of intravenously injected water soluble all-trans beta-carotene used as a food colorant.

Water soluble beta-carotene (WS-BC) is a carotenoid form that has been developed as a food colorant. WS-BC is known to contain 10% of all-trans beta-carotene (AT-BC). The aim of the present study was to investigate in vivo tissue uptake of AT-BC after the administration of WS-BC into rats. Seven-week-old male rats were administered 20 mg of WS-BC dissolved in saline by intravenous injection into the tail vein. At 0, 6, 24, 72, 120 and 168 hours (n = 7/time), blood was drawn and liver, lungs, adrenal glands, kidneys and testes were dissected. The levels of AT-BC in the plasma and dissected tissues were quantified with HPLC. After intravenous administration, AT-BC level in plasma first increased up to 6 h and returned to normal at 72 h. In the testes, the AT-BC level first increased up to 24 h and then did not decrease but was retained up to 168 h. In the other tissues, the level first increased up to 6 h and then decreased from 6 to 120 or 168 h but did not return to normal. The accumulation of WS-BC in testes but not in the other 5 tissues examined may suggest that AT-BC was excreted or metabolized in these tissues but not in testes. Although WS-BC is commonly used as a food colorant, its effects on body tissues are still not clarified. Results of the present study suggest that further investigations are required to elucidate effects of WS-BC on various body tissues.

Intraperitoneal and dietary administration of astaxanthin in rainbow trout (Oncorhynchus mykiss)--plasma uptake and tissue distribution of geometrical E/Z isomers.

Astaxanthin enters circulation in salmonid fishes upon intraperitoneal injection (IP) of small doses. Blood uptake and tissue distribution of geometrical E/Z astaxanthin isomers were determined in tissues and plasma of duplicated groups of rainbow trout (Oncorhynchus mykiss, initial weight 550 g) some of which were administered high doses of astaxanthin by IP in a trial lasting for 8 weeks. Doses of 10 (IP10), 50 (IP50) or 100 mg (IP100) astaxanthin (Lucantin Pink, BASF, Germany), respectively, dispersed in phosphate buffered saline were tested in comparison with diets containing 10 (Control) or 60 (Fed 60) mg astaxanthin kg(-1). Astaxanthin concentrations in all examined tissues and plasma were significantly higher in IP50 and IP100 than in controls and Fed 60 (p<0.05). In IP50, 11 mg astaxanthin kg(-1) muscle was detected after 4 weeks, compared to 4 mg kg(-1) in rainbow trout fed 60 mg kg(-1). Concentrations up to 80 and 100 mg astaxanthin kg(-1) were detected in liver and kidney after IP, respectively, whereas fish only fed astaxanthin contained about 2 mg astaxanthin kg(-1). No increase in muscle astaxanthin concentration was found between 4 and 8 weeks in fish given IP, and the muscle astaxanthin concentration in IP50 and IP100 were similar. Muscle concentration and injected dose were curvilinearly correlated and the proportion of ingested dose retained by the muscle was negatively correlated with the amount of injected astaxanthin. Plasma and muscle concentrations of astaxanthin were highly correlated (p<0.0001). Astaxanthin Z-isomers accumulated selectively in the various tissues after IP, whereas all-E-astaxanthin was preferably absorbed into plasma when administered via the diet. There was a selective uptake of all-E-astaxanthin in the muscle of all fish. Mortality was not affected by treatment, but a dose-dependent reduction in SGR was evident after IP. In conclusion, a more rapid and higher uptake of astaxanthin in plasma, muscle, kidney and liver of rainbow trout takes place after IP compared to when astaxanthin is fed via the diet.

[Green urine. A case report and review of the literature]. / Grüner Urin. Fallbericht und Durchsicht der Literatur.

Green urine, a well described condition, can be caused by a variety of agents. Here we describe a case of green urine caused by blue food colouring during tube feeding. In the article, we list more than 20 drugs, chemical agents and microorganisms which have been associated with green urine.

Bio-metabolic disposition of metanil yellow, orange II and their blend by caecal microflora of rats.

Caecal microflora were employed to study the degradation pattern, with time course of Metanil yellow and Orange II-two extensively used non-permitted food colours. Metanil yellow and Orange II showed the respective Degradation Index 50 (DI 50) values of 369 and 288 min. However, the blend of Metanil yellow and Orange II (1:1) resulted in the D1 50.value of 288 min. Metanil yellow, Orange II and their blend were resolved into respective metabolites in different solvent systems.

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.

Pharmacokinetic data in the evaluation of the safety of food and color additives.

Safety evaluation of food and color additives intended for human use is usually based on toxicity data obtained from animal studies; human data are rarely available. The extrapolation of animal data to humans is often controversial. The important role that pharmacokinetic data could play in the safety evaluation of food and color additives is now widely recognized. This paper reviews the current scientific knowledge concerning the application of properly designed pharmacokinetic studies to the evaluation of the safety of food and color additives. In principle, pharmacokinetic data can be useful not only in designing, interpreting, and extrapolating animal toxicity studies to humans, but also in providing insight into the mechanisms of toxicity. Examples of such applications are provided.

Evaluating the safety of food and color additives with pharmacokinetic data.

Pharmacokinetic studies are designed to quantify, as a function of time, the processes associated with absorption, distribution, metabolism, and excretion of a chemical in experimental animals or in humans. Such studies have played an important role in drug safety evaluation and could be very useful in the safety evaluation of food and color additives. This presentation provides an overview of the potential use of metabolic and pharmacokinetic data in the design and evaluation of toxicological studies and in the assessment of the potential hazard to humans from exposure to food or color additives.

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.

Metabolic disposition of 14C-labelled Brown HT in the rat, mouse and guinea-pig.

The absorption, metabolism, tissue distribution and excretion of 14C-labelled Brown HT has been studied in the rat, mouse and guinea-pig. Following administration of a single oral dose of either 50 or 250 mg Brown HT/kg, substantially all of the dose was excreted in the urine and faeces within 72 hr, with the majority (more than 80%) being accounted for in the faeces. A significant difference in urinary excretion of radioactivity was seen between male and female rats, as well as clear species differences at the two dose levels used. In all species studied, naphthionic acid was the major urinary metabolite, whereas in the faeces naphthionic acid, trace quantities of unchanged dye and at least two unidentified metabolites were found. Pregnant rats eliminated a single oral dose of 14C-labelled colouring at a rate similar to that in non-pregnant females, but some retention of radioactivity was found in the foetuses. Radioactivity was present in all tissues of male rats 24 hr after an oral dose of 250 mg 14C-labelled Brown HT/kg, with the highest concentrations in the gastro-intestinal tract, kidney and lymph nodes. Clearance from the gastro-intestinal tract was more rapid than from other tissues, but by day 7, the concentration of radioactivity (less than 0.001% of the dose/g) was similar in all tissues except the kidney and mesenteric lymph nodes. Similar results were obtained with animals pretreated for 21 days with either unlabelled or 14C-labelled Brown HT (250 mg/kg/day) prior to a radioactive dose. For most tissues examined, the concentration of radioactivity was greater with pretreatment than without. These results suggest that despite the rapid reduction and elimination of the major part of an oral dose of Brown HT, some colouring and/or metabolites accumulate in most tissues of male rats during repeated daily administration, but that only in the kidney and mesenteric lymph nodes is the accumulation tissue-specific. The accumulated radioactivity is cleared rapidly from most tissues on cessation of treatment. No significant absorption of either Brown HT, metabolites or subsidiary dyes was detected using isolated loops of small intestine.