Safety and regulatory review of dyes commonly used as excipients in pharmaceutical and nutraceutical applications

    Safety and regulatory review of dyes commonly used as excipients in pharmaceutical and nutraceutical applications Leire P´erez-Ibarbia, Tobias Majdanski, Stephanie Schubert, Norbert Windhab, Ulrich S. Schubert PII: DOI: Reference:

S0928-0987(16)30315-3 doi: 10.1016/j.ejps.2016.08.026 PHASCI 3670

To appear in: Received date: Revised date: Accepted date:

19 May 2016 20 July 2016 11 August 2016

Please cite this article as: P´erez-Ibarbia, Leire, Majdanski, Tobias, Schubert, Stephanie, Windhab, Norbert, Schubert, Ulrich S., Safety and regulatory review of dyes commonly used as excipients in pharmaceutical and nutraceutical applications, (2016), doi: 10.1016/j.ejps.2016.08.026

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Safety and Regulatory Review of Dyes Commonly Used as Excipients in

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Pharmaceutical and Nutraceutical Applications

Ulrich S. Schubertb,c,*

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Leire Pérez-Ibarbiaa, Tobias Majdanskib,c, Stephanie Schubertc,d, Norbert Windhaba,

Evonik Nutrition & Care GmbH, Kirschenallee, 64293 Darmstadt, Germany

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Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstrasse 10, 07743 Jena,

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Germany

Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany

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Institute of Pharmacy, Friedrich Schiller University Jena, Otto-Schott-Straße 41, 07743 Jena, Germany

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Corresponding author: Ulrich S. Schubert; Email: [email protected]; Phone: 0049/3641/948200

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Abstract

Color selection is one of the key elements of building a strong brand development and product identity in the pharmaceutical industry, besides to prevent counterfeiting. Moreover, colored

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pharmaceutical dosage forms may increase patient compliance and therapy enhancement. Although most synthetic dyes are classified as safe, their regulations are stricter than other classes of excipients. Safety concerns have increased during the last years but the efforts to change to natural dyes seem to be not promising. Their instability problems and the development of “non-toxic” dyes is still a challenge. This review focuses specifically on the issues related to dye selection and summarizes the current regulatory status. A deep awareness of toxicological data based on the public domain, making sure the compliance of standards for regulation and safety for successful product development is provided. In addition, synthetic strategies are provided to covalently bind dyes on polymers to possibly overcome toxicity issues. 1

ACCEPTED MANUSCRIPT Key words: dye, excipients, coloring agents, safety, regulation, toxicology, pharmaceutical,

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nutraceutical

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ACCEPTED MANUSCRIPT 1. Introduction According to Abrahart, a dye possesses color because it absorbs light in the visible spectrum

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(400 to 700 nm), has at least one chromophore (color bearing group), has a conjugated system

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(structure with single and double bonds) and exhibits resonance of electrons (Abrahart, 1977).

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If one of these characteristics is missing, the color is typically lost. This technical pragmatic definition does not exclude future developments for modern quantum matters or material based on interference physics performed on insect phenotypes. Historically, colorful

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appearance was interpreted as a sign of intended or desired pharmaceutical effects and led to

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screening discoveries. Until the middle of the nineteenth century, the coloring agents used in cosmetics, drugs and food were obtained from animals, plants and minerals. However, the discovery in 1856 of Mauveine as the first synthetic dye by William Perkin boosted the

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chemical industry, and synthetic dyes became principal constituents for food and drugs (Hunger, 2003). As an example, the fascination of P. Ehrlich on synthetic dyes with ability to

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stain and target microorganisms led him to discover Salvarsan®, an arsenic-aniline compound, which was used for the treatment of syphilis. Basing on it, the dye could be included in a

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molecule, which is toxic for the microorganism, and behaves as chemotherapeutic agent (Lloyd, Morgan, Nicholson, Ronimus & Riethmiller, 2005). For a number of years, there have been increasing concerns regarding the safety of synthetic dyes, and the issue whether natural dyes could replace synthetic dyes has been requested. Innumerous efforts have been made to extract dyes from colored plants and flowers but those attempts failed because most natural dyes are not very stable (Cristea & Vilarem, 2006). Furthermore, dyes have been strictly subjected to toxicity standards, and, as result, a number of colorants have been removed from the list of Food, Drug and Cosmetic Act (FD&C) (Barrows, Lipman & Bailey, 2003). The dyes available do not meet all the criteria required for the ideal pharmaceutical colorant. The acceptable daily intake (ADI), toxicological data and slight difference in international 3

ACCEPTED MANUSCRIPT regulations are the three key aspects determining color amount and type permitted. Previous reviews concluded the importance of the following regulatory aspects of dyes offering little

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information about data regarding toxicology (Kanekar & Khale, 2014). On the other hand,

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dye companies rarely provide information about the compliance to the regulations and

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toxicological data. Hence, the formulator has to access to data already in the public domain in order to be updated and have technical understanding for selecting an adequate color according to his need.

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However, the current research demonstrates that there is a clear evidence that dyes affect the

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behavior of children including those with food allergies, hyperactivity, AttentionDeficit/Hyperactivity Disorder (ADHD) and without any allergic or behavior disorder (Lefferts, 2016). A controversial example, Ritalin®, used for the treatment of children with

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ADHD, contains the two food dyes quinoline yellow (D&C Yellow No. 10) and fast green FCF (FD&C Green No. 3), which may exacerbate the disorder and shows that the need of a

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formulation containing safe dyes is of high priority. In addition, there have been a number of updates of the toxicological data of dyes, many of which related to the European Food

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Standards Authority (EFSA) opinions. Consequently, some of the safety threshold values have been lowered.

Therefore, this review is intended to provide, even though a palette of dyes are available worldwide with regulations, a deep awareness of toxicological data based on EFSA opinions, allowing the formulator to decide himself, which dye is suitable for each formulation and making sure the compliance of standards for regulation and safety. Furthermore, the review provides future aspects and new horizons regarding the safety of dyes for this purpose. Therefore, it is summarized that polymers with covalent bonded food dyes show improved characteristics correlating to the mentioned problems. The increase of the molar mass inhibits the adsorption by the gastro intrinsic system, which lowers or eliminates toxic effects (Dawson & Rudinger, 1975; Honohan, Enderlin, Ryerson & Parkinson, 1977; Konstantinova, 4

ACCEPTED MANUSCRIPT Cheshmedjieva-Kirkova & Konstantinov, 1999; Wang & Wingard, 1982). There are several routes to synthesize polymers bearing covalently bound food dyes (Dawson, Gless &

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Wingard, 1976; Dawson, Otteson & Davis, 1981; Dawson & Rudinger, 1975; Kaastrup &

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Sikes, 2015; Konstantinova & Bojinov, 1998). One recent example are novel polymeric-dyes,

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which can be applied as coating excipients for pharmaceuticals and nutraceuticals and demonstrate the safety of these polymers bearing covalently bound dyes (Pérez-Ibarbia, Majdanski, Schubert, Windhab & Schubert, 2016). However, the number of successful and

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applicable examples is rather limited.

2. Dye classification

Dyes may be classified according to their chemical structure or by use or by application

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method. This classification then would allow at least in principle for a structure-safety consideration including its simplifying and, thus, misleading generalizations. In the chemical

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classification, dyes are grouped according to certain common chemical structural features (chromophores) whereas the classification by application or usage is the principal system

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adopted by the Color Index (C.I.) (Society of dyers and colorists, 1971). Based on this classification, only dyes that are used for food, drugs and cosmetics will be mentioned in this review. They belong to azo, carotenoid, indigoid, quinophtalone, triarylmethane and xanthene chemical types (Hunger, 2003). The structures of the functional groups are shown in Figure 1. Azo dyes are synthetic compounds that contain two aromatic rings connected by a N=N double bond. They are the largest group of dyes including Amaranth, Sunset Yellow FCF, Tartrazine among others (Hunger, 2003). Carotenoid dyes can be natural or synthetic compounds containing long systems of double and single bonds (Bechtold & Mussak, 2009). Indigoid dyes are based on indigo. Indigo Carmine is the most commonly used dye for pharmaceutical application. Most of them are synthetic but indigo itself occurs in nature. Quinophtalone dyes are based on naphthalene-quinone structure. Quinoline Yellow is the 5

ACCEPTED MANUSCRIPT most used one, which consists of a mixture of mono- and disulfonate salts (Hunger, 2003; Podczeck & Jones, 2004). Triarylmethane dyes are synthetic organic compounds containing

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triphenylmethane backbones (Smith & Hong-Shum, 2011). Brilliant Blue FCF and Patent

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Blue are the most used in the pharmaceutical industry. Xanthene dyes are chemically based on

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a xanthene nucleus. Erythrosine is well-known for oral pharmaceutical dosage forms

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(Podczeck & Jones, 2004).

Figure 1. Schematic representation of the structures of some dyes used for coloring foods and drugs. Tartrazine (1), a synthetic lemon Yellow azo dye; Indigo Carmine (2), a blue indigoid dye; Quinoline Yellow (3), a quinophtalone dye; Erythrosine (4), a xanthene dye; Betacarotene (5), one of the most commonly used natural carotenoid; Brilliant Blue FCF (6), a triarylmethane dye commonly used for food, drugs and cosmetics.

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3. Rationale for coloring of pharmaceuticals and nutraceuticals with dyes

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Coloring agents or colorants are excipients used in the pharmaceutical industry to impart color

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to pharmaceutical dosage forms (Abrahart, 1977). Coloring agents can be either dyes or

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pigments. The difference between them is solubility. Natural and organic dyes are soluble in the medium in which they are applied, whereas pigments are insoluble, staying suspended in a liquid (Zollinger, 2003). Numerous coloring agents are used in pharmaceutical manufacturing

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to provide products a distinctive, identifiable appearance and to impart a uniform and

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attractive color. For example, many commercial medicaments do not differ optically and may confuse the patients leading to medical errors. Therefore, the more different is the size, shape and color of pharmaceutical dosage forms, the easier will be the identification of medications

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(Swarbrick, 2006). This distinctive appearance may help particularly those elderly patients with difficulties to read the label of the packages, ensuring an appropriate intake.

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Another important issue is the relation between counterfeit and medication errors, which increased the last years (Rodomonte, Gaudiano, Antoniella, Lucente, Crusco & Bartolomei,

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2010). Since most of the tablets are round and white and, consequently, easier to fake, a unique and distinctive color can improve the identification but makes difficult the counterfeiting. FDA works strongly on developing guidelines for the industry to help preventing from these fakes (U.S. Food and Drug Administration U.S., Department of Health & Human Services, 2009). Beside the aesthetic appearance and patient compliance, the color of the product may also have an influence on the efficacy of the therapy due to the psychological effect. The color red means "exciting", "active", "hot", "dangerous", blue is rather "calm", "cool" and "relaxed" while white color is associated with "pure," "undefiled" and "neutral". For instance, sedatives are therefore often blue colored, stomachic drugs green, strong painkillers and cardiovascular drugs red, antidepressants and stimulants rather red, yellow or pastel colors and contraceptive 7

ACCEPTED MANUSCRIPT pills lavender or pink (de Craen, Roos, de Vries & Kleijnen, 1996). Another significant benefit for the pharmaceutical industry is the use of color for brand development and product

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identity. For example, think blue and Viagra® immediately comes to mind and think purple

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think on Nexium®.

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Different problems associated with its manufacturing and use may arise when choosing an appropriate dye for a specific pharmaceutical dosage form. The application of dyes requires an understanding and knowledge of the composition of the products to be colored. A common

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problem using colors in pharmaceutical formulation is the stability (Delgado-Vargas &

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Paredes-López, 2003). As mentioned before, dyes are water-soluble and exhibit their color by being dissolved in a solvent, but in the presence of other agents like oxidizing and reducing agents, strong acids and alkalis, excessive heating and light exposure, dyes may lose their

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original color (Nicholson & Tucker, 1960). Therefore, the dye has to be chemically stable in the presence of the other ingredients and should not interfere with other agents. For example,

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synthetic dyes exhibit a good stability to heat in comparison to natural dyes. Interestingly, several FD&C dyes exhibit a rather good stability to low pH values than to high pH values.

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Under oxidizing and reducing agents, dyes show rather low stability. However, it is clear that it is not sufficient to have color formulations with better stability. Consequently, the abovementioned information must be used in combination with other strategies. These strategies include purity criteria, safety and toxicological aspects for individual colorants. Additionally, different hues may be obtained by blending primary synthetic colors. The synthetic colors that may be used to obtain secondary colors are FD&C Blue #1, FD&C Blue #2, FD&C Red #3, FD&C Red #40, FD&C Yellow #5 and FD&C Yellow #6 (Marmion, 1991). Synthetic dyes are mainly used in their water-soluble form but depending on the formulation, they may be used in the form of insoluble lakes (Wou & Mulley, 1988). Aluminum lakes are produced by precipitating and adsorbing a water soluble dye onto a water insoluble substrate, typically aluminum hydroxide. Approximately up to 10% dyes are added to formulations 8

ACCEPTED MANUSCRIPT whereas pigments are added up to 20%. In this section, the most common colored pharmaceutical dosage forms will be mentioned:

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3.1. Tablets

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Dyes may be incorporated into tablets by adding them directly as solution during wet

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granulation process or by most conventional manner adding them to a coating formulation, which is applied onto the tablet surface. However, many difficulties (e.g. mottling, fastness, migration, low degree of color uniformity) can arise relating to the dye solubility (Felton &

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McGinity, 2008). Therefore, the use of pigments is the most extended method for coloring

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tablets. 3.2. Capsules

Capsules are mainly colored with dyes that are listed as FD&C and D&C. The color is

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therefore added to the gelatin melt (Podczeck & Jones, 2004). One of the major factors to take into account is the pH value of the gelatin because it can alter the shade of the color.

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3.3. Pharmaceutical syrups

The dyes for this purpose should be completely soluble in the particular solvent. Factors like

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pH value, microbial activity, light exposure and compatibility with other ingredients should be considered because they may influence the shade and stability of the dyes. Normally, the dyes chosen correspond to the flavor of the product (red with strawberry, yellow with lemon etc.). The dye concentration in liquid preparations should be between 0.0005% and 0.001% (Troy, Remington & Beringer, 2006).

4. International regulatory status Due to frequent regulation changes, it is difficult to maintain the list of coloring agents updated for pharmaceutical and nutraceutical use. Moreover, for this purpose, dyes are more strictly regulated than other excipients, and the legislation varies from country to country. Colors that are accepted in some countries are banned in others. 9

ACCEPTED MANUSCRIPT The European Commission (EC) in the European Union (EU); the Food and Drug Administration (FDA) in the United States; as well as Ministry of Health, Labour and Welfare

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in Japan and Government of Canada in Canada are among the authorities responsible for

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defining and maintaining specifications and for the assessment of the safety in different

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applications (European Union, 2012a; Hallagan, Allen & Borzelleca, 1995). In many regions, regulators distinguish between coloring agents that may be used in drug products and those that are permissible in food. Moreover, dye names may vary, however, the Color Index (CI)

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numbers are the same. An interesting example, which evidences this fact, is the difference

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between regulations concerning Quinoline Yellow in Europe and D&C Yellow #10 in the United States. Same Color Index, same appearance but listed for different uses. Where D&C Yellow #10 is approved for drugs and cosmetics in U.S., Quinoline Yellow is approved in

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Europe for use in food and drugs. However, Japan does not approve it for use at all in foods or

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drugs (U.S. Food and Drug Administration, 2015c).

4.1. Purity requirements

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In addition, dyes have to comply with standards of purity that are required to protect the consumer. (Set out in Commission Directive 2008/128/EC laying down specific purity criteria concerning colors for use in foodstuffs). The purity specifications for color additives approved in Europe are laid down in European Directive 95/45/EC (European Union, 1995). Usually, a dye is a mixture, and the specifications include limits on the quantities of other substances which are allowed to be present. For example, simple organic salts may be present but are not hazardous. However other substances such as non-sulfonated amines, which may be present in the initial raw materials, must be eliminated as far as possible.

4.2. Dye regulation in the European Union

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ACCEPTED MANUSCRIPT Most European countries follow the European Directives that list the dyes and specifications for use in foods and drugs in the EU. The directive that has previously controlled the

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approved dyes for use in pharmaceuticals in Europe is 78/25/EEC, which is based on

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colorants approved to use in foods listed by the European Directive 94/36/EC in 1994

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(European Union, 1995). In the past years, some modifications were introduced; e.g. the color Red 2G was delisted in 2007 because of safety concerns (European Union, 2007). The EU system assigns to all approved dyes which are defined as food additives, an “E” number.

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Regulation (EC) No 1331/2008 of the European Parliament and the Council of 16 December

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2008 established a common authorization procedure for food additives, food enzymes and food flavorings (European Union, 2008). Commission Regulation (EU) No 231/2012 of 9 March 2012 laid down specifications for food additives listed in Annexes II and III to

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Regulation (EC) No 1333/2008 of the European Parliament and of the Council. These are described in the Official Journal of the European Union. The dyes approved by the EU are

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listed in the Table 1.

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4.3. Dye regulation in the United States The FDA is responsible to regulate the list of dyes intended to use in the United States. These dyes must comply with chemical specifications, uses, restrictions and labeling requirements described in the Code of Federal Regulations (CFR) in Title 21, parts 70-82. The regulations for the purity are specified in the 21 CFR Part 74 and Part 82. The FDA divided the color additives in two categories: The one that are subject to certification processes including azo, xanthene, triarylmethane and indigoid dyes (originally from coal-tar colors and nowadays mainly synthetized from oil) and the other exempt from certification, including the color additives derived from natural sources, like ß-carotene, which are not listed in Table 1 (U.S Food and Drug Administration, 2012).

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ACCEPTED MANUSCRIPT When a color additive is intended for a new application, a petition should be made to the FDA. In the case that the petition is approved, the FDA will publish a new list (Food and

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Drug Administration U.S Department of Health and Human Services 2012). The FDA act

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provides that food, drugs, cosmetics and devices are considered adulterated if they have not

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been proved safe for their use. Moreover, the color additive certification assures that the freshly manufactured batches of color additives comply with the specification requirements and identity (Barrows, Lipman & Bailey, 2003). This certification is continually reviewed and

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updated according to toxicological findings for certified colors. These changes are the

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removal of certification, the transfer of a color additive from one category to another and the addition of new colors to the list. In addition, the pharmaceutical manufacturer is responsible to ensure that the dyes comply with the specifications. Comparable to the EU System, the

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responsibility for assuring the quality of the dyes is the responsibility of the manufacturer.

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Dyes subject to certification and permanently listed by FDA are shown in Table 1.

4.4. Dye regulation in Canada

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In Canada, regulations for dyes are similar to the U.S. regulations. Most of the colors listed for use in United States and in Europe are listed in Canada as well but some that are approved in United States are banned in Canada and vice versa. Approved dyes, which can be used in Canadian drugs, are listed in Table 1 (Government of Canada, Research Centre Communication 2009).

4.5. Dye regulation in Japan In Japan, the Japanese Ministry of Health, Labor and Welfare (MHLW) is responsible for the approval of color additives, which are outlined in a positive list of approved colors, including their regulations. Synthetic dyes permitted in Japan include dyes approved in the EU or/and in

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ACCEPTED MANUSCRIPT the U.S. (Delgado-Vargas & Paredes-López, 2003). Approved dyes in Japan are shown in

Color Name

Approved in

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Acid Fuchsine

US, CA

45100

Acid Red

JP

13058

Alba Red

US

61570

Alizarin Cyanine Green

US, CA

60730

Alizarin Violet

US

60725

Alizurol Purple SS

US

16035

Allura Red AC

EU, US,

16035:1

Allura Red AC

EU, US

FD&C Red #40 Lake

42090

Alphazurine FG

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D&C Blue #4

16185

Amaranth

28440

E129

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E123

Brilliant Black BN

EU

E151

42090

Brilliant Blue FCF

EU, US, CA

E133

20285

Brown HT

EU

E155

14720

E122

Red #106 D&C Red #39 D&C Green #5

Ext. D&C Violet #2

FD&C Red #40

Red #2

FD&C Blue #1

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EU, JP

74160

D&C Red #33

D&C Violet #2

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17200

12156

Japanese Name

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Number

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Color Index

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Table 1.

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Citrus Red #2

Copper Phthalocyanine

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[Phthalocyaninato (2-)]

Carmoisine

Blue #1

Copper 45370:1

Dibromofluorescein

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D&C Orange #5

45425:1

Diiodofluorescein

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D&C Orange #10

45380

Eosine

US, CA

D&C Red #22

45430

Erythrosine

EU, US, CA, JP

42053

Fast Green FCF

US, CA, JP

FD&C Green #3

12085

Flaming Red

US, CA

D&C Red #36

45350:1

Fluorescein

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D&C Yellow #7

E127

FD&C Red #3

Red #3

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Green S

EU

E142

73360

Helindone Pink CN

US, CA

D&C Red #30

73000

Indigo

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D&C Blue #6

73015

Indigotine

EU, US, CA, JP

15850

Lithol Rubin B

US, CA

15850:1

Lithol Rubin B Ca

US, CA

10316

Napthol Yellow S

US

15510

Orange II

US

42051

Patent Blue V

EU

45410

Phloxine B

US, CA, JP

16255

Ponceau 4R

EU, CA, JP

14700

Ponceau SX

US, CA

FD&C Red #4

59040

Pyranine Concentrated

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D&C Green #8

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Orange B

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D&C Green #6

FD&C Blue #2

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D&C Red #6

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D&C Red #7

Ext. D&C Yellow #7 D&C Orange #4

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E131

D&C Red #28

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E124

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19235

Blue #2

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E132

Red #104(1) Red #102

Quinizarine Green SS

47005

Quinoline Yellow

20170

Resorcin Brown

45440

Rose Bengal

JP

15985

Sunset Yellow FCF

EU, US, CA, JP

E110

FD&C Yellow #6

Yellow #5

19140

Tartrazine

EU, US, CA, JP

E102

FD&C Yellow #5

Yellow #4

45380:2

Tetrabromo Fluorescein

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D&C Red #21

45410:1

Tetrachlorotetra-

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D&C Red #27

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61565

EU, US

E104

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D&C Yellow #10 D&C Brown #1 Red #105(1)

Bromofluorescein 26100

Toney Red

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D&C Red #17

45350

Uranine

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D&C Yellow #8

Table 1 List of dyes approved by EU, United States, Canada and Japan EU1= European Union; US2= United States; CA3= Canada; JP4= Japan

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ACCEPTED MANUSCRIPT Approved dyes listed by the EU. The above list is from Part B, List of All Additives, from Annex II to Regulation (EC) No 1333/2008 on food additives. Although the original list contains both pigments and dyes, here are only dyes listed. 2

Approved dyes listed by FDA. List of Permanently Listed dyes Subject to Batch Certification. Listed in CFR Title 21. Although the original

list contains both pigments and dyes, here are only dyes listed for use in foods and drugs.

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List of approved dyes for internal use in Japan. These dyes are approved by Ministry of Health, Labour and Welfare (MHLW).

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List of dyes approved for use in drugs in Canada.

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5. Toxicological aspects of dyes

In drug formulation, the safety of excipients is as important as the safety of the active product

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ingredient. For new excipients, thorough evaluation is required by the EMA and FDA for the excipient itself (U.S. Food and Drug Administration 2005). However, there is not a formal

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procedure for approval of single new excipients. Excipients are approved when the new drug

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formulation containing the novel excipient receives regulatory approval. Therefore, some

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manufacturers do not risk the possibility that FDA may reject a new formulation containing also a new excipient. The experimental, non-clinical toxicological evaluation of the separate

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excipient is expensive as well as time-consuming but as the safety of the patients is of highest priority, the evaluation is a necessity. The Ecological and Toxicological Association of the Dyestuff Manufacturing Industry (ETAD), an online database on publications of toxicological effects and legislation concerning dyes (and pigments), identifies and assesses of the acute and chronic toxicological effects caused by dyes (Nicholson & Tucker, 1960). The European Food Standards Authority (EFSA) is responsible for assessing the safety of synthetic food, drugs and cosmetic dyes in Europe. The “Acceptable Daily Intake” (ADI) approach started in 1961(Lu, 1988). The need for a toxicological evaluation of each substance led to the Joint FAO/WHO Expert Committee

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ACCEPTED MANUSCRIPT on Food Additives (JECFA) experts to establish a dosage level that causes no significant effects in animals and humans. The definition of ADI was discussed and clarified, and in 1987

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the ADI term was reported. The definition was expressed as follows: Amount of substance (in

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this case dye) expressed on a body weight basis that can be consumed daily over a lifetime

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without appreciable health risk (Food and Agriculture Organization of the United Nations & World Health Organization, 1987).

Among the synthesized dyes, azoic dyes are included in pharmaceutical formulations due to

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their excellent coloring properties. But on the other hand, they are well known for causing

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some adverse effects. A study demonstrated that their reduction by intestinal microflora causes the formation of aromatic amines. The intermediates and end products have been seen to be carcinogenic and mutagenic (Chung, 1983). The azo dye Tartrazine (FD&C Yellow No.

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5) is known to be dangerous in aspirin-intolerant patients. Nonetheless, reactions to Tartrazine are similar to those produced by aspirin, occurring in patients both with and without a history

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of aspirin intolerance (Virchow Szczeklik, Bianco, Schmitz-Schumann, Juhl & Robuschi, et al., 1988). Urticaria was the first evidence found by Lockey followed by an association

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between asthma caused by ingestion of Tartrazine and asthmatic patients with previous aspirin intolerance (Lockey, 1959). Beside allergic reactions, hyperactivity has been evidenced as well (McCann, Barrett, Cooper, Crumpler, Dalen & Grimshaw, 2007). Challenge studies were carried out basing that exposures to low levels of dyes could cause rapid deterioration in the behavior of children. In 2007, a study was conducted by the University of Southampton (“Southampton study”) in the United Kingdom (U.K.). The study was intended to investigate whether certain color additive mixtures and the widely used preservative, sodium benzoate, when consumed in a beverage, cause hyperactivity in three-year-old and eight- and nine-year-old children. The investigated colors were Tartrazine, Ponceau 4R, Sunset Yellow FCF, Carmoisine, Quinoline Yellow and Allura Red (For C.I. Number see Table 1). EFSA examined the results of the above 16

ACCEPTED MANUSCRIPT mentioned study and concluded that there is evidence that certain mixtures of dyes tested had a small but statistically significant effect on activity and attention in children (European Food

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Safety Authority, 2009a). Consequently, EFSA lowered the ADIs of Quinoline Yellow,

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Sunset Yellow and Ponceau 4R whereas the ADIs of Tartrazine, Carmoisine and Allura Red

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remained unchanged. However, the meta-analyses published after the Southampton study demonstrated that the ADHD symptoms have been improved with a diet free of food dyes for children with and without behavior disorder, which indicates that ADI values set by EFSA

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indeed may cause behavioral disorders. A study on the reproductive toxicity of Erythrosine in

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male mice showed that the administration of Erythrosine in doses of 680 and 1360 mg/kg/bw/d has a potential toxic effect on spermatogenesis in mice (Aziz, Shouman, Attia, & Saad, 1997). In 1990, the FDA removed Erythrosine from the list for use in

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cosmetics and externally applied drugs and the lakes for use in food, drug, and cosmetic products because it may induce cancer in rats (U.S. Food and Drug Administration, 2015a).

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If some of the medicines contain the above-described dyes, these dyes require labeling. Recently, the regulation 21 CFR 201.20 was published, where those drug products intended

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for human use administered orally, nasally, rectally or vaginally and in the area of the eye containing FD&C Yellow 5 (Tartrazine) and FD&C Yellow 6 (Sunset Yellow) require labeling. This is due to the potential to cause allergic reactions (even bronchial asthma) in susceptible persons and in those who have aspirin hypersensitivity (U.S. Food and Drug Administration, 2015b). Furthermore, the FDA and the Canadian government showed that the levels of bound benzidine (carcinogenic contaminant) found in Tartrazine and Sunset Yellow, exceeded levels of free dyes (Lancaster & Lawrence, 1991; Peiperl, Prival, & Bell, 1995). A compilation of the most frequently reported toxicological effects of the dyes included in this review and their ADI values are shown in Table 2.

17

ACCEPTED MANUSCRIPT

Classification/

ADI

Chemical class

(mg/kg/bw)

Allergic reactions

Hyperactivity

Reproductive

Genotoxicity

Carcinog

toxicity

and/or

enicity

respiratory tract irritation

children in

(mainly those patients with

combination

asthma and aspirin

with other dyes

MA N

intolerance)

No adverse

Biological

No

(Committee on Drugs,

effects up to

significance of

potential

1997; European Food

levels of 773 and

the positive

to induce

Safety Authority, 2009g;

1225

genotoxicity

benign or

Hallagan, Allen &

mg/kg/bw/day for

results in other

malignan

Borzelleca, 1995; Tanaka,

males and females

studies is

t

Takahashi, Oishi &

respectively

uncertain

neoplasia

Ogata, , 2008)

CR

Possible for

US

May cause eye, skin, and

TE D

7.5

mutagenicity

s

CE P

Synthetic/Azo

Ref.

AC

Tartrazine

IP

T

Dye

18

ACCEPTED MANUSCRIPT

Classification/

ADI

Chemical class

(mg/kg/bw)

Allergic reactions

Hyperactivity

Reproductive

Genotoxicity

Carcinog

toxicity

and/or

enicity

children in

when Quinoline Yellow is

combination

taken as part of a mixture

with other dyes

with other synthetic colors)

Ref.

mutagenicity

A study in rats

In vivo and in

Levels

(Committee on Drugs,

provides rationale

vitro assays

up to

1997; European Food

for reevaluating

(cellular

2500

Safety Authority, 2009e;

the ADI

models) showed

mg/kg/b

Macioszek &

potential

w/day in

Kononowicz, 2004)

mutagenicity

the rat

CR

rhinitis and asthma (mostly

US

Possible for

MA N

Quinophtalone

May cause urticaria,

TE D

Yellow

0.5

CE P

Synthetic/

AC

Quinoline

IP

T

Dye

and 7500 mg/kg/b w/day in the mouse revealed no evidence of carcinog enicity

19

ACCEPTED MANUSCRIPT

Classification/

ADI

Chemical class

(mg/kg/bw)

Allergic reactions

Hyperactivity

Reproductive

Genotoxicity

Carcinog

toxicity

and/or

enicity

intolerance reaction

children in

(particularly amongst those

combination

with an aspirin

with other dyes

MA N

intolerance)

mutagenicity

No tumor

In vitro data

No

(Committee on Drugs,

induction

indicate direct-

effects

1997; European Food

acting oxidative

on tumor

Safety Authority, 2009f;

genotoxicity

formatio

Sweeney, Chipman &

may be induced

n

Forsythe, 1994)

CR

Possible for

US

May cause urticaria and/or

TE D

1

CE P

Yellow

Synthetic/Azo

Ref.

by reaction products of azo dyes (including Sunset Yellow)

AC

Sunset

IP

T

Dye

20

ACCEPTED MANUSCRIPT

Classification/

ADI

Chemical class

(mg/kg/bw)

Allergic reactions

Hyperactivity

Reproductive

Genotoxicity

Carcinog

toxicity

and/or

enicity

urticaria

combination

MA N

with other dyes

Several studies

No critical

Anticarci

(European Food Safety

with no adverse

genotoxicity

nogen

Authority, 2010a, 2011;

effect

Maldonado-Cervantes,

CR

Possible in

US

Few isolated cases of

TE D

0.15

mutagenicity

effects in rat

Jeong, León-Galván, Barrera-Pacheco, De León-Rodríguez & González de Mejia, 2010)

CE P

Synthetic/Azo

Ref.

AC

Amaranth

IP

T

Dye

21

ACCEPTED MANUSCRIPT

Classification/

ADI

Allergic reactions

Chemical class

(mg/kg/bw)

Hyperactivity

Reproductive

Genotoxicity

Carcinog

toxicity

and/or

enicity

0.7

Possible in

cross-reaction with some

combination

other material)

with other dyes

mutagenicity Unlikely to

Studies

(European Food Safety

on reproduction

induce any

with

Authority, 2009d;

significant

Ponceau

European Union, 2012b;

genotoxic risk

4R do

Tanaka, 2006)

not show any

TE D e

Xanthene

No evidence 0.1

CE P

Synthetic/

AC

Erythrosin

Ref.

No adverse effect

MA N

4R

Sensitization (due to a

CR

Synthetic/Azo

US

Ponceau

IP

T

Dye

carcinog enic effect

Effects seen on

Potential toxic

In vitro

Rodent

(European Food Safety

behavior

effect in

genotoxicity but

thyroid

Authority, 2011;

changes

spermatogenesis

not in vivo

cancer,

Kobylewski & Jacobson,

returned to

not in

2010)

normal levels

humans

within next 7 hours post dose

22

ACCEPTED MANUSCRIPT

Classification/

ADI

Allergic reactions

Chemical class

(mg/kg/bw)

Hyperactivity

Reproductive

Genotoxicity

Carcinog

toxicity

and/or

enicity

Possible in

reactions (mostly taken

combination

within mixtures of other

with other dyes

synthetic colors) Carmoisin

Synthetic/Azo 4

mutagenicity Unlikely to

Neoplas

(European Food Safety

incidence of

induce any

ms in

Authority, 2009b)

fetuses

significant

rats

genotoxic risk

No sensitizing activity (in

In combination

High doses in rats

Unlikely to

No

(Bär & Griepentrog,

experiments on guinea-

with mixtures of

produced bladder

induce any

evidence

1960; European Food

hyperplasia

significant

of

Safety Authority, 2009c)

genotoxic risk

carcinog

TE D

e

Ref.

Increase on the

CR

7

Urticarial and vasculitis

US

Synthetic/Azo

MA N

Allura Red

IP

T

Dye

pigs, it was found that

other dyes

CE P

Azorubine/Carmoisine had no skin sensitizing

AC

activity)

enicity

Patent

Synthetic/

Blue V

Triarylmethane

Anaphylatic reactions 5

No data

No evidence of

No concern

No

(European Food Safety

available

reproductive

regarding

evidence

Authority, 2013)

toxicity

genotoxicity

of carcinog enicity

23

ACCEPTED MANUSCRIPT

Classification/

ADI

Allergic reactions

Chemical class

(mg/kg/bw)

Hyperactivity

Reproductive

Genotoxicity

Carcinog

toxicity

and/or

enicity

Carmine

2.5

May cause respiratory

No data

problems if inhaled,

available

Blue FCF

Triarylmethane

Skin irritation

No data

TE D

Synthetic/ 6

available

mutagenicity No data

No

(Mancuso, Staffa, Errani,

effects on

available

evidence

Berdondini & Fabbri,

of

1990)

reproduction

carcinog enicity No signs of

No concern

No

(European Food Safety

toxicity

regarding

evidence

Authority, 2010b; U.S.

genotoxicity

of

Food and Drug

carcinog

Administration 2005)

CE P

Brilliant

MA N

irritant to skin and eyes

Ref.

No adverse

CR

Synthetic/Indoid

US

Indigo

IP

T

Dye

AC

enicity in rats or mice

Betacarotene

Natural/Carotenoid

No adverse effects 5

No adverse

No adverse

No adverse

No

(Bechtold & Mussak,

effects

effects

effects

adverse

2009)

effects Table 2 Health effects of the most frequently used dyes for pharmaceuticals and nutraceuticals

24

ACCEPTED MANUSCRIPT Although dyes are frequently used in food and drug formulations for decades, the evaluation of their toxicological potential is still ongoing based on new findings. Additional data are

T

required to determine if human exposure to dyes results in irreversible adverse health effects.

IP

Regarding hyperactivity studies, the majority of them are being conducted on children

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described as hyperactive or with a clinical diagnosis of ADHD. As a consequence, from 2004 up to now, three meta-analyses have been conducted. In 2004, the first one concluded that there is a strong association between ingestion of food dyes and hyperactivity (Schab & Trinh,

NU

2004). The meta-analyses from 2012 (Nigg, Lewis, Edinger & Falk, 2012) and 2013 (Sonuga-

MA

Barke et al., 2013) demonstrated that the ADHD symptoms improved with a diet free of food dyes for children with and without behavior disorder. In consequence, the British government led to protect the public by encouraging the elimination of certain food dyes. The European

TE

D

Parliament wanted to inform the public about the risks of dyes with a warning labeling on most dyed foods sold in the EU. In contrast to these actions, the FDA acknowledged that the

AC

problems.

CE P

use of dyes might affect the behavior of some children with ADHD and other behavior

6. Dye labeled polymers for overcoming toxicity One strategy to overcome toxicity issues is the chemical immobilization of possibly critical food dyes onto polymeric carriers. Such polymeric dyes might show reduced interaction with cells compared to the free dyes. In detail, the dye immobilization is of high importance in particular for gastro intestinal applications, since some food dyes are genotoxic or cause allergic reactions. Thus, the use of polymeric food dyes avoids the uptake of the pure dyes by the gastro intestinal system (Dawson & Rudinger, 1975). It could further be shown that polymeric food dyes are more stable towards light than the single food dyes. The routes to covalently bind FDA approved dyes to a polymer are as various as their applications (Figure 2). The dye is either employed in a pure manner or modified chemically. In case of chemical 25

ACCEPTED MANUSCRIPT modification, small molecules are bound to the FDA approved dye to obtain the functionality needed or the dye is bound to a polymer directly to label the polymer structure or to add other

T

characteristics. Beside the benefits of polymer food dyes, also disadvantageous properties due

IP

to the dye modification are reported. Dynapol® for example observed slight color deviations

SC R

between the polymer food dye and the non-bound form. To overcome this problem is a challenging issue and led to termination of further developments and tests in some cases (Furia, 1980). In the following part, we report on several different methods to bind food dyes

NU

to polymers. It must be noted that this part strongly focuses on the synthesis, and the products

AC

CE P

TE

D

MA

are not always meant to be used as food additives.

Figure 2. Possible modification strategies of polymers with FDA approved dyes. a) Nanotube modified Eosin as anionic initiator, b) monomer modified with an FDA approved dye, c) overview of functional groups on dyes and polymers that are used for post modification 26

ACCEPTED MANUSCRIPT reactions, d) synthetic route invented by Dynapol® to obtain FDA food dye colored

T

poly(acrylic acid).

IP

Besides the obvious application some of the FDA approved dyes are also used as radical

SC R

promoters like Eosin and Rose Bengal. Consequently, they can also be used as anionic initiator as shown for Eosin (Chen, Anbarasan, Kuo & Chen, 2011). A further strategy is the modification of a FDA approved dye with a monomer that can be used in polymerizations

NU

(Blas, Stelzer & Slugovc, 2006; Konstantinova, Cheshmedjieva-Kirkova & Konstantinov,

MA

1999; Konstantinova & Bojinov, 1998; Konstantinova & Venkova, 2006). Already prepared polymers can also be applied for the synthesis of polymeric FDA approved dyes via post-modification in a direct manner (Kaastrup & Sikes, 2015; Nakanishi, Satoh, Norisuye &

TE

D

Tran-Cong-Miyata, 2004). For this purpose, it is sometimes necessary to modify the dye with a spacer, which enables a reaction with the polymer side chain or end group (Lee & Sikes,

CE P

2014). Dynapol® invented another method where FDA approved food dyes were bound to polymers by synthesizing the dye directly at the polymer side chain via azo coupling

AC

(Dawson, Gless & Wingard, 1976; Dawson & Rudinger, 1975). In the following, we focus on FDA approved dyes, which show strong absorbance in the visible light range. Consequently, the wide and already fully established field of fluorescein labeling is out of focus of this review.

6.1. FDA approved dye initiator The only report about a FDA dye as initiator is published by P.H. Chen et al. for poly(εcaprolactone) synthesis (Chen, Anbarasan, Kuo & Chen, 2011). For this purpose, the carboxylic acid of Eosin Y was modified with microwalled carbonnanotubes (MWCNT); the product was applied for the initiation of ε-caprolactone. The aromatic hydroxyl group of the

27

ACCEPTED MANUSCRIPT Eosin Y scaffold served as initiating group. The product is a ε-caprolactone with a single

T

MWCNT-Eosin Y unit suitable for photodynamic therapy (Figure 2a).

IP

6.2. FDA approved dye monomers

SC R

Similar to the already mentioned Eosin modification with multiwall carbon nanotubes MWCNT, the carboxyl group can also be used for modifications with monomers (Figure 2b). The reaction is most often a substitution at the dye with the monomer compound like a

NU

norbornene derivative. The obtained monomer was polymerized via ring-opening metathesis

MA

polymerization. The product was afterwards used for the formulation of pH sensitive dyed polymer particles (Blas, Stelzer & Slugovc, 2006).

D

Another example contains the functionalization of Eosin with vinylbromide. The resulting

TE

product is a bifunctional Eosin with a vinylether and a methacrylvinyl unit. The vinyl- and acryl-modified dye was added during the polymerization to obtain the dyed poly(vinyl ether)

CE P

and poly(acryl amide), respectively (Konstantinova & Venkova, 2006). Even though the application of this dye as food and cosmetic additive is mentioned in the literature, the focus

AC

of the research concentrated on the synthesis and the spectroscopic and photochemical properties. No further toxicological tests are described within this work. A similar route is described by Liu et al.. Herein, the use of 4-vinylbenzylchloride as modification monomer (Figure 2b) was applied. The obtained monomer was polymerized by reversible addition-fragmentation chain transfer (RAFT) using different acrylic co-monomers. The resulting polymers are used for the fabrication of photosensitizer-conjugated nanocarriers with pH switchable phototherapy modules (Guhuan, Jinming, Guoying, & Shiyong, 2015).

6.3. Polymeric FDA approved dyes by post-modification For post-modifications of polymers with FDA approved dyes, the polymer and/or the dye need to have suitable functional groups, which are able to react efficiently. Modifications of 28

ACCEPTED MANUSCRIPT the side chains are most commonly used. H.D. Sikes et al. used commercially available isocyanate modified Eosin for the post modification of dendrimers. The isocyanate was then

T

directly reacted with the end standing amines of the dendrimers, yielding a product used for

IP

macrophotoinitiator applications (Kaastrup & Sikes, 2015). A further example shows an

SC R

isocyanine Eosin derivate and a semi Boc-protected ethylene diamine, which leads to an Eosin with a primary amine group after cleaving the protection group (Figure 2c). The resulting product was used for post-modification of poly(acrylic acid) via EDC coupling that

NU

can be further used for radical initiations with Eosin (Lee & Sikes, 2014). EDC coupling was

MA

also applied for the labeling of gelatin with amine side chains, which could react with Eosin in a direct manner. The obtained product serves as a cross-linker, which is suitable for dental applications (Fukaya, Nakayama, Murayama, Omata, Ishikawa, Hosaka & Nakagawa, 2009).

TE

D

As already mentioned, in situ synthesis of azo food dyes at the polymer side chain are also described in several patents released by Dynapol®. For the synthesis of these compounds, a

CE P

poly(acrylic acid) is partially modified via Schmidt rearrangement, where the carboxyl side groups are transferred into amines under strong acidic conditions to obtain a copolymer of

AC

acrylic acid and vinylamine (Figure 2d). The copolymer is then reacted with selected sulfochlorides to obtain polymeric sulfonanilamides, which are (after hydrolysis) used for the azo coupling reaction. Consequently, poly(acrylic acid) with food dyes like Sunset Yellow, Tartrazine and Amaranth are obtained. The resulting products are called Poly RTM-481 (Amaranth), Poly YTM-607 (Tartrazine) and Poly RTM-478 (Sunset Yellow). This method is limited to azo food dyes and comes along with several synthesis steps (Dawson, Gless & Wingard, 1976; Dawson & Rudinger, 1975).

6.4. Toxicological studies

Since most of the mentioned food dye modifications for the synthesis of food dye containing polymers were not intended to be used as food additives, no tests regarding toxicity or 29

ACCEPTED MANUSCRIPT intestinal absorption for the explicit modifications were found in literature. This is not valid for the post-modified polymers reported by Dynapol® being synthesized with the aim to enter

T

the market. The resulting products underwent several tests induced by Dynapol® and other

IP

researching groups. In these tests, different issues such as metabolism, adsorption, and

SC R

mutagenicity were examined. Adjusted designs of tests of the adsorbed dyes and metabolites were performed with excretions, carbon dioxide, blood test etc.. For this purpose, the formulations were labeled with 14C isotopes to detect the increased radioactivity. Furthermore,

NU

thin layer chromatography was performed in some cases to give quantitative predictions of the

MA

tested probes. It was found that the azo bond stability is critical under in vivo conditions and could not be improved significantly by the polymer attachment (Honohan, Enderlin, Ryerson & Parkinson, 1977; Walson, Carter, Ryerson, Clark & Parkinson, 1981). In contrast, a high

TE

D

stability of the sulfonamide bond could be determined. For the intact polymeric food dye, almost no intestinal adsorption was observed as well as a decrease of sulfanilic acid, which

CE P

leads to the assumption that the sulfonamide bond is stable under in vivo conditions. This observation confirms that the concept of polymeric food dyes to reduce intestinal adsorption

AC

is principally working. A detailed review of the physical, chemical and biological properties as well as a chronological overview of the polymer food dyes development process induced by Dynapol® is given by T. E. Furia (Furia, 1980; Parkinson Brownt &, 1985).

7. Conclusions and future prospects Dyes are important excipients in pharmaceutical formulations, which can be used for enhancing the aesthetic appearance, product differentiation, and, most importantly, they may reduce errors in medication. The list of approved dyes is limited and while certified dyes are still viewed with a note of mystery, their safety may be assured due to the complex regulatory involved. As the toxicity of a given dye will always be related to the dose, some dyes that are generally known to be “non-toxic” are in fact toxic to humans in large doses. In addition, an

30

ACCEPTED MANUSCRIPT increasing number of safety concerns during the last decades led a substantial need for the development and approval of new “non-toxic” dyes. As safety studies are time-consuming

T

and expensive, possibly more acceptable dyes by consumers would be those of natural origin

IP

such as carotenoids. However, the drawback with these colors is that they are unstable, their

SC R

coloring power is usually lower, and they may show many incompatibilities. Moreover, the lack of information and management could be improved by compiling a database to identify the problems of pharmaceuticals related to dyes, regulations and safety

NU

strategies. Regulations may vary from country to country but ADI values and toxicological

MA

data are the same worldwide, thus confirming the need of an international database. That would provide a better understanding of the use, approval and risk of these dyes by formulators and consumers.

TE

D

Future developments in the field of reducing dye toxicity by dye chemical modification processes are promising and more research is recommended, including a comprehensive

CE P

updated exposure assessment. For the above reasons, it is important that the formulator takes into consideration regulatory and toxicology issues associated with the selection of dyes as

AC

well as the technical and formulation aspects relating to their successful incorporation into pharmaceutical dosage forms. Regarding patients, the groups at risk to be considered with attention are people with allergies and intolerances.

8. Acknowledgements We acknowledge funding from the Carl-Zeiss Foundation (JCSM Strukturantrag) and the Thüringer Ministerium für Wirtschaft, Wissenschaft und Digitale Gesellschaft (TMWWdG, ProExzellenzΙI, NanoPolar).

Conflict of interest The authors have no conflict of interest to declare. 31

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Abrahart, E.N. (1977). Dyes and their intermediates. (2nd Revised ed.). Lincoln: Hodder Arnold Aziz, A. A. H., Shouman, S. A., Attia, A. S., & Saad, S. F. (1997). A study on the reproductive toxicity of Erythrosine in male mice. Pharmacological Research, 35, 457-462. Bär, F., Griepentrog, F. (1960). Die Allergenwirkung von fremden Stoffen in Lebensmitteln. Medizin und Ernährung, 1, 99-104. Barrows, J.N., Lipman, A.L., & Bailey, C.J. (2003). Color Additives: FDA's Regulatory Process and Historical Perspectives. Food Safety Magazine. Bechtold, T., & Mussak, R. (2009). Handbook of natural colorants. In U. G. Chandrika, Carotenoid dyes – Properties, (pp. 221-236). John Wiley & Sons, Ltd. Blas, M., Stelzer, F., & Slugovc, C. (2006). Covalent incorporation of Eosin into ROM-polymers and preparation of polymer particles. Polymer Preprints, 47, 499. Brownt, J.P, & Parkinson, T.M. (1985). Nonabsorbable Food Additives through Polymeric Design. Drug Metabolism Reviews, 16, 389-422. Chen, H.-H., Anbarasan, R., Kuo, L.-S., & Chen, P.-H. (2011). A novel report on Eosin Y functionalized MWCNT as an initiator for ring opening polymerization of ɛ-caprolactone. Materials Chemistry and Physics, 126, 584-590. Chung, K.-T. (1983). The significance of azo-reduction in the mutagenesis and carcinogenesis of azo dyes. Mutation Research/Reviews in Genetic Toxicology, 114, 269-281. Committee on Drugs. (1997). “Inactive” ingredients in pharmaceutical products: Update. Pediatrics, 99, 268-278. Cristea, D., & Vilarem, G. (2006). Improving light fastness of natural dyes on cotton yarn. Dyes and Pigments, 70, 238-245. Dawson, D. J,.Gless, R. D., & Wingard, R. E. (1976). Poly(vinylamine hydrochloride). Synthesis and utilization for the preparation of water-soluble polymeric dyes. Journal of American Chemical Society, 98, 5996-6000. Dawson, D.J., Otteson, K.M., & Davis, R. (1981). Polymeric yellow colorant. US Patent 4,250,327, (10.02.1981). Dawson, D. J., & Rudinger, J. (1975). Food containing non-toxic food coloring compositions and a process therefor. US Patent 3,920,855, (18.11.1975). de Craen, A. J. M., Roos, P. J, de Vries, A. L., & Kleijnen, J. (1996). Effect of colour of drugs: systematic review of perceived effect of drugs and of their effectiveness. British Medical Journal, 313, 1624-1626. Delgado-Vargas, F., & Paredes-López, O. (2003). Natural colorants for food and nutraceutical uses. Boca Raton: CRC Press (Chapter 5). European Food Safety Authority. (2009a). http://www.efsa.europa.eu/en/press/news/ans091112 Accessed 12.02.2016. European Food Safety Authority. (2009b). Scientific opinion on the re-evaluation of Allura Red AC (E 129) as a food additive. European Food Safety Authority Journal. European Food Safety Authority. (2009c). Scientific opinion on the re-evaluation of Azorubine/Carmoisine (E 122) as a food additive. European Food Safety Authority Journal. European Food Safety Authority. (2009d). Scientific opinion on the re-evaluation of Ponceau 4R (E 124) as a food additive. European Food Safety Authority Journal. European Food Safety Authority. (2009e). Scientific opinion on the re-evaluation of Quinoline Yellow (E 104) as a food additive. European Food Safety Authority. European Food Safety Authority. (2009f). Scientific opinion on the re-evaluation of Sunset Yellow FCF (E 110) as a food additive. European Food Safety Authority Journal. European Food Safety Authority. (2009g). Scientific opinion on the re-evaluation Tartrazine (E 102) EFSA panel on food additives and nutrient sources added to food (ANS). European Food Savety Authority Journal. European Food Safety Authority. (2010a). Scientific opinion on the re-evaluation of Amaranth (E 123) as a food additive. European Food Safety Authority Journal. 32

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European Food Safety Authority. (2010b). Scientific opinion on the re-evaluation of Brilliant Blue FCF (E 133) as a food additive. European Food Safety Authority Journal. European Food Safety Authority. (2011). Scientific opinion on the re-evaluation of Erythrosine (E 127) as a food additive. European Food Safety Authority Journal. European Food Safety Authority. (2013). Scientific opinion on the re-evaluation of Patent Blue V (E 131) as a food additive. European Food Safety Authority Journal. European Union. (1995). Commission Directive 95/45/EC of 26 July 1995 laying down specific purity criteria concerning colors for use in foodstuffs (Official Journal of the European Union 26.7.1995, L206, p.1). European Union. (2007). Commission regulation (EC) No. 884/2007 on emergency measures suspending the use of E 128 Red 2G as food colour. Official Journal of the European Union, (L 195, 27 July 2007, pp. 8/9). European Union. (2008). Regulation (EC) No 1334/2008 of the European Parliament and of the council of 16 December 2008 on flavourings and certain food ingredients with flavouring properties for use in and on foods and amending council regulation (EEC) No 1601/91, regulations (EC) No 2232/96 and (EC) No 110/2008 and directive 2000/13/EC. Official Journal of the European Union, (L 354, 31.12.2008, p. 34–50.). European Union. (2012a). Commision regulation (EU) No 231/2012 of 9 March 2012 laying down specifications for food additives listed in Annexes II and III to regulation (EC) No 1333/2008 of the European Parliament and of the council. Journal of the European Union, (L83, 9.3.2012, 1295). European Union. (2012b). Commission regulation (EU) No 232/2012 of 16 March 2012 amending Annex II to regulation (EC) No 1333/2008 of the European Parliament and of the council as regards the conditions of use and the use levels for Quinoline Yellow (E 104), Sunset Yellow FCF/Orange Yellow S (E 110) and Ponceau 4R, Cochineal Red A (E 124). Official Journal of the European Union, (L 78, 16.3.2012, 1-12.). Felton, L. A., & McGinity, J. W. (2008). Aqueous polymeric coatings for pharmaceutical dosage forms. In Nyamweya, N.N., & Hoag, S.W. (Eds.), Influence of coloring agents on the properties of polymeric coating systems). CRC Press. Food and Agriculture Organization of the United Nations & World Health Organization (1987). Technical Report Series of World Health Organization, Evaluation of certain food additives and contaminants., U.S. Food and Drug Administration , Department of Health and Human Services (2012). Food and drugs. Chapter I. Subchapter A. General. Fukaya, C., Nakayama, Y., Murayama, Y., Omata, S., Ishikawa, A., Hosaka, Y., & Nakagawa, T. (2009). Improvement of hydrogelation abilities and handling of photocurable gelatin-based crosslinking materials. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 91B, 329-336. Furia, T. (1980). CRC Handbook of Food Additives,. (2nd ed.). Taylor & Francis Government of Canada, Research Centre Communication (2009). Food and drug regulations. C.R.C c. 870, Last amended on November 7,2014. Guhuan, L., Jinming, H., Guoying, Z., & Shiyong, L. (2015). Rationally Engineering Phototherapy Modules of Eosin-Conjugated Responsive Polymeric Nanocarriers via Intracellular Endocytic pH Gradients. Bioconjugate Chemistry, 26, 1328-1338. Hallagan, J. B., Allen, D. C., & Borzelleca, J. F. (1995). The safety and regulatory status of food, drug and cosmetics colour additives exempt from certification. Food and Chemical Toxicology, 33, 515-528. Honohan, T., Enderlin, F. E., Ryerson, B. A., & Parkinson, T. M. (1977). Intestinal absorption of polymeric derivatives of the food dyes Sunset Yellow and Tartrazine in rats. Xenobiotica, 12, 765-774. Hunger, K. (2003). Industrial dyes: chemistry, properties, applications. (3rd ed.). Wiley-VCH

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