Using High-Throughput Screening to Evaluate Perturbations Potentially Linked to Neurobehavioral Outcomes: A Case Study Using Publicly Available Tools on FDA Batch-Certified Synthetic Food Dyes.

There is growing evidence from human and animal studies indicating an association between exposure to synthetic food dyes and adverse neurobehavioral outcomes in children. However, data gaps persist for potential mechanisms by which the synthetic food dyes could elicit neurobehavioral impacts. We developed an approach to evaluate seven US FDA-batch-certified food dyes using publicly available high-throughput screening (HTS) data from the US EPA's Toxicity Forecaster to assess potential underlying molecular mechanisms that may be linked to neurological pathway perturbations. The dyes were screened through 270 assays identified based on whether they had a neurological-related gene target and/or were mapped to neurodevelopmental processes or neurobehavioral outcomes, and were conducted in brain tissue, targeted specific hormone receptors, or targeted oxidative stress and inflammation. Some results provided support for neurological impacts found in human and animal studies, while other results showed a lack of correlation with in vivo findings. The azo dyes had a range of activity in assays mapped to G-protein-coupled receptors and were active in assays targeting dopaminergic, serotonergic, and opioid receptors. Assays mapped to nuclear receptors (androgen, estrogen, and thyroid hormone) also exhibited activity with the food dyes. Other molecular targets included the aryl hydrocarbon receptor, acetylcholinesterase, and monoamine oxidase. The Toxicological Prioritization Index tool was used to visualize the results of the Novascreen assays. Our results highlight certain limitations of HTS assays but provide insight into potential underlying mechanisms of neurobehavioral effects observed in in vivo animal toxicology studies and human clinical studies.

Improvement of Naturally Derived Food Colorant Performance with Efficient Pyranoanthocyanin Formation from Sambucus nigra Anthocyanins Using Caffeic Acid and Heat.

Consumers and regulations encourage the use of naturally derived food colorants. Anthocyanins (ACN), plant pigments, are unstable in foods. In aged red wines, ACN with a free hydroxyl group at C-5 condenses to form pyranoanthocyanins (PACN), which are more stable but form inefficiently. This study attempted to produce PACN efficiently using high cofactor concentration and heat. Elderberry anthocyanins were semi-purified and caffeic acid (CA) was dissolved in 15% ethanol and diluted with a buffer to achieve ACN:CA molar ratios of 1:50, 1:100, 1:150, and 1:200, then incubated at 65 °C for 5 days. The effect of temperature was tested using ACN samples incubated with or without CA at 25 °C, 50 °C, and 75 °C for 7 days. Compositional changes were monitored using uHPLC-PDA-MS/MS. Higher CA levels seemed to protect pigment integrity, with ACN:CA 1:150 ratio showing the highest tinctorial strength after 48 h. PACN content growth was fastest between 24 and 48 h for all ACN:CA ratios and after 120 h, all ACN had degraded or converted to PACN. PACN formed faster at higher temperatures, reaching ~90% PACN in 24 h and ~100% PACN in 48 h at 75 °C. These results suggest that PACN can form efficiently from elderberry ACN and CA if heated to produce more stable pigments.

Dietary exposure assessment of synthetic food colours using analytical concentrations in Korea.

The safety of nine synthetic colour additives approved in Korea were assessed through hazard identification, hazard characterisation, exposure assessment, and risk characterisation. Hazard identification and characterisation were conducted using new risk information based on toxicology data. Exposure assessment calculated the estimated daily intake (EDI) of the nine colours. Finally, the risk was evaluated by comparing the EDIs of the colours with the acceptable daily intake (ADI). 1,114 samples (24 food categories) among a total of 1,454 samples contained colour additives either at a level that was not detectable or up to a quantified maximum of 288 mg kg-1. The likelihood of risk of exposure to all food colours, evaluated by comparing the EDI with the ADI, was less than 0.52% of the ADI for the general population. These results indicate that use of synthetic food colour in Korea is safe and does not result in excessive exposure to any population group.

Preparation and Characterization of Acylcaramel.

Caramel is a widely used water-soluble food pigment. The acylation of caramel was conducted by aliphatic acyl chlorides with different chain lengths. Acetyl, butyryl, octyl, lauryl, palmityl, and stearyl caramels were prepared with the ratio of acyl chloride/caramel of 6. The formation of acylated caramel was confirmed by Fourier transform infrared spectra, and the acyl mass fraction in acylcaramel was determined by potentiometric titration. Thermal analysis showed that the weight loss of acylated caramel was higher than that of raw caramel. The scanning electron microscopy analysis showed that the morphology of acylated caramel was significantly different from that of raw material. The acyl mass fraction of acylated caramel increased with the increase of acyl chain lengths. Meanwhile, the lipo-hydro partition coefficient, the solubility in corn oil, and color, red, and yellow indexes increased with the increase of the mass fraction of acyl in acylcaramel. It was found that stearyl caramel has the highest lipid solubility of 5.73 mg/mL in corn oil; however, the color, red, and yellow indexes of palmityl caramel reached 25 818.60, 1.149, and 1.757, respectively. This study provides a method to improve the solubility of caramel in lipid phase and expand the application range of caramel.

Synthesis of different metallochlorophyllins and quantification in food samples by reversed phase - high performance liquid chromatography.

The complex formation between metals (Ni, Co, Zn, Fe, Pb, Sn and Ag) and natural chlorophyll extracted from green leaves was monitored in the present study. The respective metallochlorophyllin was prepared by the reaction of metal chloride or nitrate (1M) to chlorophyll extracted from Ficus leaves extract. All synthesized metallochlorophyllins were stable and Na-Cu-chlorohyllin (E141) which is permitted to add in food and are listed in European Directive 94/36/EC on food colouring materials, was identified in commercially available food commodities (candies). In this study different synthesized metallochlorophyllins were characterised by using UV-Vis, FT-IR, HPLC, AAS and HR-MS techniques. Many food commodities (i.e. candy, chips, drink, and cream biscuits) were monitored for metallochlorophyllins and Na-Cu-Chlorophyllin was detected only in real candy samples.

Ultra-High-Throughput Analytical Strategy Based on UHPLC-DAD in Combination with Syringe Filtration for the Quantitation of Nine Synthetic Colorants in Beverages: Impacts of Syringe Membrane Types and Sample pH on Recovery.

An ultra-high-throughput approach based on ultra-high-performance liquid chromatography with diode array detection (UHPLC-DAD) in combination with simple syringe filtration was successfully developed and validated for the quantitation of nine synthetic colorants in beverages. The recoveries of the colorants from the beverages were found to be dramatically affected by the syringe filter membrane types and pH of the sample solution. The high recoveries of the nine colorants (92.7-105.9%) were achieved by syringe filtration with poly(vinylidene difluoride) membrane following the pH adjustment of sample solution at pH 7.0. The sample treatment procedure was very simple and took only 1 min. The fast chromatographic separation (1 min) of the nine synthetic colorants was achieved by UHPLC-DAD using a C18-core-shell column. This analytical approach (UHPLC-DAD combined with syringe filtration) took only approximately 3 min. The established method was ultrafast, sensitive, precise, accurate, and reliable. The method was successfully applied to rapidly determine the 9 colorants in 17 beverages.

Identification of the monobrominated derivative of Acid Red 52 (Food Red No. 106) in pickled vegetables.

Two unknown dyes (purple and purplish-red) were detected by TLC in two pickled vegetable (sakura-zuke daikon) samples containing Acid Red 52 (AR) and New Coccine as food colorants. HPLC with diode-array detection and LC/MS analyses suggested that the purple dye is monobrominated AR and the purplish-red dye is its N-desethyl derivative, which would be generated mainly during sample preparation. For the identification of the purple dye, a reference compound was prepared by bromination of AR followed by isolation of the monobrominated AR, the structure of which was elucidated as 4'-brominated AR (4'BrAR) by LC/ToF-MS and (1)H-NMR spectroscopy. The purple dye was confirmed as 4'BrAR by comparison of its retention time, ultraviolet-visible spectrum and mass spectrum with those of the prepared reference compound. To our knowledge, this is the first report of the detection of 4'BrAR in foods.

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.

Effect of ascorbic acid on the properties of ammonia caramel colorant additives and acrylamide formation.

Ammonia caramels are among the most widely used colorant additives in the food industry. They are commonly prepared through the Maillard reaction and caramelization of mixtures of reducing sugars with ammonia or ammonium salts. Antioxidants are known to inhibit acrylamide formation during the Maillard reaction, and they may affect the properties of the ammonia caramel products. Thus, the objective of this study was to investigate the effect of the antioxidant ascorbic acid on the properties of ammonia caramel. A mixture of glucose and ammonia was allowed to react at 120 °C for 60 min in the presence of ascorbic acid at final concentrations of 0 to 0.08 M. The ammonia caramels obtained from these reactions were all positively charged. As the concentration of ascorbic acid increased, the color intensity of the ammonia caramel showed a decreasing trend, while the intensity of the fluorescence and total amount of pyrazines in the volatiles showed a tendency to increase. The addition of ascorbic acid did not result in obvious changes in the UV-visible spectra of the ammonia caramels and the types of pyrazines in the volatiles were also unchanged. It is noteworthy that the addition of 0.02 to 0.08 M ascorbic acid did reduce the formation of the by-product acrylamide, a harmful substance in food. When the concentration of ascorbic acid added reached 0.04 M, the content of acrylamide in the ammonia caramel was 20.53 µg/L, which was approximately 44% lower than that without ascorbic acid. As a result, ascorbic acid can be considered to improve the quality and safety of ammonia caramels.

Filamentous fungi are large-scale producers of pigments and colorants for the food industry.

With globalization in the research trends, healthier life styles, and the growing market for the natural food colorants in the economically fast-growing countries all over the world, filamentous fungi are being investigated as readily available sources of chemically diverse colorants. With two selected examples, polyketide-Monascus-like pigments from the new fungal production strains, and the promising and yet unexplored hydroxy-anthraquinoid colorants, the present review highlights exciting recent findings, which may pave the way for alternative and/or additional biotechnological processes for the industrial production of natural food colorants of improved functionality. As an additional aspect, marine fungi are discussed as potential sources of novel pigments of numerous color hues and atypical chemical structures.

Biotechnological production of colorants.

The color of food and drinks is important, as it is associated with freshness and taste. Despite that natural colorants are more expensive to produce, less stable to heat and light, and less consistent in color range, natural colorants have been gaining market share in recent years. The background is that artificial colorants are often associated with negative health aspects. Considerable progress has been made towards the fermentative production of some colorants. Because colorant biosynthesis is under close metabolic control, extensive strain and process development are needed in order to establish an economical production process. Another approach is the synthesis of colors by means of biotransformation of adequate precursors. Algae represent a promising group of microorganisms that have shown a high potential for the production of different colorants, and dedicated fermentation and downstream technologies have been developed. This chapter reviews the available information with respect to these approaches.

Optimised formation of blue Maillard reaction products of xylose and glycine model systems and associated antioxidant activity.

BACKGROUND: A blue colour can be formed in the xylose (Xyl) and glycine (Gly) Maillard reaction (MR) model system. However, there are fewer studies on the reaction conditions for the blue Maillard reaction products (MRPs). The objective of this study is to investigate characteristic colour formation and antioxidant activities in four different MR model systems and to determine the optimum reaction conditions for the blue colour formation in a Xyl-Gly MR model system, using the random centroid optimisation program. RESULTS: The blue colour with an absorbance peak at 630 nm appeared before browning in the Xyl-Gly MR model system, while no blue colour formation but only browning was observed in the xylose-alanine, xylose-aspartic acid and glucose-glycine MR model systems. The Xyl-Gly MR model system also showed higher antioxidant activity than the other three model systems. The optimum conditions for blue colour formation were as follows: xylose and glycine ratio 1:0.16 (M:M), 0.20 mol L⁻¹ NaHCO3, 406.1 mL L⁻¹ ethanol, initial pH 8.63, 33.7°C for 22.06 h, which gave a much brighter blue colour and a higher peak at 630 nm. CONCLUSION: A characteristic blue colour could be formed in the Xyl-Gly MR model system and the optimum conditions for the blue colour formation were proposed and confirmed.

Synthesis and application of (13)C-labeled 2-acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI), an immunosuppressant observed in caramel food colorings.

2-Acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI) is a minor toxic contaminant observed in caramel food colorings and was shown to exert immunosuppressant activity when fed to rodents. Because of this toxicity, maximum levels of THI in caramel food colorings have been defined by international and European authorities. Several reports of THI analysis using external standardization have been published for liquid foods such as beers and soft drinks. However, no suitable internal standard has yet been described allowing THI analysis in more complex samples. In this paper we describe the preparation of a labeled [(13)C6]THI analogue and its application for the successful validation of the first stable isotope dilution assay (SIDA) of THI in caramel food colorings. A brief survey of THI levels in commercially available caramel class III (E 150c) and IV (E 150d) food colorings is also included, corroborating that THI occurs only in caramel class III food colorings.

A rapid shaking-based ionic liquid dispersive liquid phase microextraction for the simultaneous determination of six synthetic food colourants in soft drinks, sugar- and gelatin-based confectionery by high-performance liquid chromatography.

A novel and simple rapid shaking-based method of ionic liquid dispersive liquid phase microextraction for the determination of six synthetic food colourants (Tartrazine, Amaranth, Sunset Yellow, Allura Red, Ponceau 4R, and Erythrosine) in soft drinks, sugar- and gelatin-based confectionery was established. High-performance liquid chromatography coupled with an ultraviolet detector was used for the determinations. The extraction procedure did not require a dispersive solvent, heat, ultrasonication, or additional chemical reagents. 1-Octyl-3-methylimidazolium tetrafluoroborate ([C8MIM][BF4]) was dispersed in an aqueous sample solution as fine droplets by manual shaking, enabling the easier migration of analytes into the ionic liquid phase. Factors such as the [C8MIM][BF4] volume, sample pH, extraction time, and centrifugation time were investigated. Under the optimum experimental conditions, the proposed method showed excellent detection sensitivity with limits of detection (signal-to-noise ratio=3) within 0.015-0.32 ng/mL. The method was also successfully used in analysing real food samples. Good spiked recoveries from 95.8%-104.5% were obtained.

Determination of seven synthetic dyes in animal feeds and meat by high performance liquid chromatography with diode array and tandem mass detectors.

An efficient method was developed for the simultaneous determination of seven commonly used synthetic sulfonate dyes (Ponceau 4RC, Sunset yellow, Allura red, Azophloxine, Ponceau xylidine, Erythrosine and Orange II) in animal feed and meat using high performance liquid chromatography (HPLC-DAD) and tandem mass spectrometry (HPLC-MS/MS). Ethanol-ammonia-water (80:1:19, V/V/V) solution was used as extract solution, which can extract target species while reducing interference from the sample matrices. The recoveries of these 7 dyes in animal feed and chicken meat were between 71% and 97% with relative standard deviations less than 14.8%. HPLC-MS/MS was employed as a further means of confirmation to assure accuracy of the results. Limits of detection for these dyes were in the range of 0.02-21.83 ng mL(-1). The proposed method can be applied to confirmative screening of seven commonly used food colorants in feed and meat samples.

Carcinogenicity and regulation of caramel colorings.

2- and 4-methylimidazoles are present as contaminants in caramel colorings manufactured with ammonia catalysts. Both contaminants have been shown to induce cancer in animals and may be present in caramel colorings in amounts that exceed federal guidelines. California requires warning notices on products that could lead to consumption of more than 30 micrograms per day. The US Food and Drug Administration should bar the use of excessively contaminated caramel coloring in food.

Modeling of chromatographic lipophilicity of food synthetic dyes estimated on different columns.

The retention behavior of some food synthetic dyes has been studied by RP-HPLC on chemically bonded C18, C8, C16 and CN stationary phases. Using methanol-ammonium acetate (0.08 mol/L, pH=6.76) as mobile phase, a linear behavior of retention parameters throughout the methanol fraction variance was obtained in all cases (r>0.99). The patterns of chromatographic behavior of the compounds illustrate high similarities between the C18, C8 and C16 columns, respectively. Highly significant correlations were obtained between experimental lipophilicity indices log k(w) and phi(0) estimated on C18 and C8 stationary phases and some computed log P-values. An extensive investigation made for quantitative structure-property (lipophilicity) relationships of studied dyes, using descriptors from Dragon software, multiple linear regression and genetic algorithm, revealed that the molecular descriptors appearing in the best models combine 2-D and 3-D aspects of the molecular structure. The most significant descriptors can be classified as radial distribution function, GETAWAY (autocorrelation), 3D-MoRSE signal, Burden eigenvalues and edge adjacency descriptors.

A review of chromatographic methods for determination of synthetic food dyes.

The purpose of this work was to present a chromatographic methods to analyse synthetic food dyes. The following techniques has been described: thin-layer liquid chromatography (TLC), high performance thin-layer chromatography (HPTLC), traditional column chromatography, high performance liquid chromatography (HPLC), include: ion-pair chromatography (HPLC IP), reversed phase chromatography (RP HPLC) and high performance ion chromatography.

Determination of 13 synthetic food colorants in water-soluble foods by reversed-phase high-performance liquid chromatography coupled with diode-array detector.

A reversed-phase high performance liquid chromatographic method for the successful separation and determination of 13 synthetic food colorants (Tartrazine E 102, Quinoline Yellow E 104, Sunset Yellow E 110, Carmoisine E 122, Amaranth E 123, Ponceau 4R E 124, Erythrosine E 127, Red 2G E 128, Allura Red AC E 129, Patent Blue V E 131, Indigo Carmine E 132, Brilliant Blue FCF E 133 and Green S E 142) was developed. A C18 stationary phase was used and the mobile phase contained an acetonitrile-methanol (20:80 v/v) mixture and a 1% (m/v) ammonium acetate buffer solution at pH 7.5. Successful separation was obtained for all the compounds using an optimized gradient elution within 29 min. The diode-array detector was used to monitor the colorants between 350 and 800 nm. The method was thoroughly validated. Detection limits for all substances varied between 1.59 (E 142) and 22.1 (E 124) microg L(-1). The intra-day precision (as R.S.D.(r)) ranged from 0.37% (E 122 in fruit flavored drink at a concentration of 100 mg L(-1)) to 4.8% (E 142 in icing sugar at a level of 0.9 mg kg(-1)). The inter-day precision (as R.S.D.(R)) was between 0.86% for E 122 in fruit flavored drink at 100 mg L(-1) and 10% for E142 in jam at a concentration of 9 mg kg(-1). Satisfactory recoveries, ranging from 94% (E 142 in jam) to 102% (E 131 in sweets), were obtained. The method was applied to the determination of colorants in various water-soluble foods, such as fruit flavoured drinks, alcoholic drinks, jams, sugar confectionery and sweets, with simple pre-treatment (dilution or water extraction).

Monitoring of the generation of non-mutagenic benzoic acid compounds during the manufacture of erythrosine.

The generation of 2-(2',4'-dihydroxybenzoyl) benzoic acid (DHBBA) and 2-(2',4'-dihydroxy-3',5'-diiodobenzoyl) benzoic acid (DHDIBBA) during the manufacture of erythrosine (Food Red No. 3) was examined. DHBBA is formed as an intermediate during the synthesis of fluorescein, and as fluorescein is produced, it is gradually consumed. However, under inappropriate reaction conditions, it remains in the resulting fluorescein at the termination of synthesis. DHDIBBA is easily obtained by the iodination of DHBBA. These compounds are also found when erythrosine is heated in excessive alkali. The results of the Ames test using DHBBA and DHDIBBA showed they did not possess mutagenic activity. The results clearly demonstrated that establishment of an upper limit for DHBBA and DHDIBBA is important in the quality control of fluorescein and erythrosine.