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EFSA Journal 2012;10(5):2675

SCIENTIFIC OPINION

Scientific Opinion on the safety and efficacy of Allura Red AC (E 129) in feed for cats and dogs1
EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3
European Food Safety Authority (EFSA), Parma, Italy

ABSTRACT
Allura Red AC is intended to be used as a colourant in feedingstuffs for cats and dogs without quantitative limitations. Since the additive is only applied for non-food-producing animals, the assessment of safety is limited to the target species and the user. The FEEDAP Panel considered that (i) genotoxicity of Allura Red AC cannot be excluded; (ii) the mouse carcinogenicity study may not be appropriate for investigating the risk of colon cancer that might result from local DNA damage; and (iii) Allura Red AC is proposed for lifetime use in cats and dogs, leading to a much higher exposure in target animals than in humans. The FEEDAP Panel concluded that the available data are insufficient to demonstrate the safety of Allura Red AC for cats and dogs. In the absence of any information, the substance should be considered to be potentially harmful as a result of skin, eye, or inhalation exposure by users of the additive. Its colouring efficacy in a typical complementary feed for dogs was demonstrated at a minimum dose of 50 mg/kg feed. Allura Red AC is considered efficacious in adding colour to feed for cats and dogs. © European Food Safety Authority, 2012

KEY WORDS
Allura Red AC, sensory additive, colourant, cat, dog, safety

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On request from the European Commission, Question No EFSA-Q-2011-00214, adopted on 24 April 2012. Revision 1 – 21 May 2012: minor editorial changes. Panel members: Gabriele Aquilina, Georges Bories, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Noël Albert Dierick, Mikolaj Antoni Gralak, Jürgen Gropp, Ingrid Halle, Christer Hogstrand, Reinhard Kroker, Lubomir Leng, Secundino López Puente, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Giovanna Martelli, Miklós Mézes, Derek Renshaw, Maria Saarela, Kristen Sejrsen and Johannes Westendorf. Correspondence: FEEDAP@efsa.europa.eu Acknowledgement: The Panel wishes to thank the members of the Working Group on Colouring agents, including Annette Schuhmacher, for the preparatory work on this scientific opinion.

Suggested citation: EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP); Scientific Opinion on the safety and efficacy of Allura Red AC (E 129) in feed for cats and dogs. EFSA Journal 2012;10(5):2675. [14 pp.] doi:10.2903/j.efsa.2012.2675. Available online: www.efsa.europa.eu/efsajournal

© European Food Safety Authority, 2012

Allura Red AC (E129) for cats and dogs

SUMMARY
Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver an opinion on the safety and efficacy of Allura Red AC (E 129) in feed for cats and dogs. Allura Red AC is already approved for feed and food use. Allura Red AC is intended to be used as a colourant in feedingstuffs for cats and dogs without quantitative limitations. Since the additive is only applied for non-food-producing animals, the assessment of safety is limited to the target species and the user. The FEEDAP Panel considered that: (i) genotoxicity of Allura Red AC cannot be excluded; (ii) the mouse carcinogenicity study may not be appropriate for investigating the risk of colon cancer that might result from local DNA damage; and (iii) Allura Red AC is proposed for lifetime use in cats and dogs, leading to a much higher exposure in target animals than in humans. The FEEDAP Panel concluded that the available data are insufficient to demonstrate the safety of Allura Red AC for cats and dogs. In the absence of any information, the substance should be considered to be potentially harmful as a result of skin, eye, or inhalation exposure by users of the additive. The colouring efficacy of Allura Red AC in a typical complementary feed for dogs was demonstrated at a minimum dose of 50 mg/kg feed. Allura Red AC is considered efficacious in adding colour to feed for cats and dogs.

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Allura Red AC (E129) for cats and dogs

TABLE OF CONTENTS
Abstract ....................................................................................................................................................1 Summary ..................................................................................................................................................2 Table of contents ......................................................................................................................................3 Background ..............................................................................................................................................4 Terms of reference....................................................................................................................................5 Assessment ...............................................................................................................................................7 1. Introduction ......................................................................................................................................7 2. Characterisation ................................................................................................................................7 2.1. Particle size ...............................................................................................................................8 2.2. Manufacturing process ..............................................................................................................8 2.3. Stability and homogeneity ........................................................................................................8 2.4. Conditions of use ......................................................................................................................9 2.5. Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) 9 3. Safety ................................................................................................................................................9 3.1. Safety for the target animals .....................................................................................................9 3.1.1. Genotoxicity studies including mutagenicity ...................................................................9 3.1.1.1. In vitro tests ............................................................................................................. 9 3.1.1.2. In vivo tests ............................................................................................................ 10 3.1.2. Conclusions from the genotoxicity tests.........................................................................10 3.1.3. Long-term studies ...........................................................................................................10 3.1.4. Conclusion ......................................................................................................................11 3.2. Safety for the user ...................................................................................................................11 4. Efficacy ..........................................................................................................................................11 Conclusions ............................................................................................................................................12 Recommendation ....................................................................................................................................12 Documentation provided to EFSA .........................................................................................................12 References ..............................................................................................................................................12 Appendix ................................................................................................................................................14

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Allura Red AC (E129) for cats and dogs

BACKGROUND
Regulation (EC) No 1831/20034 establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular, Article 4(1) of that Regulation lays down that any person seeking authorisation for a feed additive or for a new use of a feed additive shall submit an application in accordance with Article 7. In particular Article 10(2) of that Regulation also specifies that for existing products within the meaning of Article 10(1), an application shall be submitted in accordance with Article 7, at the latest one year before the expiry date of the authorisation given pursuant to Directive 70/524/EEC5 for additives with a limited authorisation period, and within a maximum of seven years after the entry into force of this Regulation for additives authorised without time limit or pursuant to Directive 82/471/EEC.6 The European Commission received a request from the company Feed Additives Synthetic Colours Group7 for re-evaluation of the product Allura Red AC (E 129), when used as a feed additive for cats and dogs (category: 2. sensory additive; functional group: (a) colourants/substances that add or restore colour in feedingstuff) under the conditions mentioned in Table 1. According to Article 7(1) of Regulation (EC) No 1831/2003, the Commission forwarded the application to the European Food Safety Authority (EFSA) as an application under Article 4(1) (authorisation of a feed additive or new use of a feed additive) and under Article 10(2) (re-evaluation of an authorised feed additive). EFSA received directly from the applicant the technical dossier in support of this application.8 According to Article 8 of that Regulation, EFSA, after verifying the particulars and documents submitted by the applicant, shall undertake an assessment in order to determine whether the feed additive complies with the conditions laid down in Article 5. The particulars and documents in support of the application were considered valid by EFSA as of 22 July 2011. Allura Red AC (E 129) is included in the European Union Register of Feed Additives pursuant to Regulation (EC) No 1831/2003. It is authorised without a time limit in application of Article 9t (b) of Council Directive 70/524/EEC concerning additives in feedingstuffs (2004/C 50/01) for its use in cats and dogs as colourant additive (colouring agents authorised for colouring foodstuffs by Community rules). The additive is also authorised for all species or categories of animals with the exception of cats and dogs for animal feedingstuffs only in products processed from: (i) waste products of foodstuffs, (ii) other base substances, with the exception of cereals and manioc flour, denaturated by means of these agents or coloured during technical preparation to ensure the necessary identification during manufacture. No maximum levels of Allura Red AC in feeds are established in the EU. The applicant asks for the re-evaluation of the use of the additive for cats and dogs. Allura Red AC is an approved food colourant in the EU and it is listed in Annex I of Directive 94/36/EC9 of 30 June 1994. According to the same Directive Allura Red AC is authorised as synthetic food colouring substance in the EU, with a maximal allowed use level of 25-500 mg/kg food for various foodstuffs. Allura Red AC is also allowed in alcoholic beverages at levels up to 200 mg/L and non-alcoholic beverages up to 100 mg/L.

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Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition. OJ L 268, 18.10.2003, p. 29. Directive 70/524/EEC of 23 November 1970 concerning additives in feeding-stuffs. OJ L 270, 14.12.1970, p.1. Council Directive 82/471/EEC of 30 June 1982 concerning certain products used in animal nutrition. OJ L 213, 21.07.1982, p.8. Feed Additives Synthetic Colours Group. Oldmedow Road, PE30 4LA, Kings Lynn, UK. EFSA Dossier reference: FAD-2010-0347. European Parliament and Council Directive 94/36/EC of 30 June 1994 on colours for use in foodstuffs. OJ L 237, 10.09.1994, p. 13.

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The specific purity criteria concerning the use of Allura Red AC in foodstuffs are included in Commission Directive 2008/128/EC.10 Allura Red AC has been evaluated previously by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1974 (JECFA, 1974), 1980 (JECFA, 1980) and 1981 (JECFA, 1981) and the Scientific Committee for Food (SCF) in 1975 (EC, 1975), 1984 (EC, 1984) and 1989 (EC, 1989). Allura Red AC was evaluated in 2000 by the National Toxicological Program (NTP, 2000). In 2002, the Nordic Working Group on Food Toxicology and Risk Assessment (NNT, 2002) reviewed the current status and safety data on all food additives permitted in the EU, including Allura Red AC. The EFSA Panel on food additives and nutrient sources added to food (ANS) issued a scientific opinion on the re-evaluation of Allura Red AC as a food additive (EFSA, 2009). In 2010 the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) issued a scientific opinion on the appropriateness of the food azo-colours (including also Allura Red AC) for inclusion in the list of food ingredients set up in Annex IIIa of Directive 2000/13/EC (EFSA, 2010).

TERMS OF REFERENCE
According to Article 8 of Regulation (EC) No 1831/2003, EFSA shall determine whether the feed additive complies with the conditions laid down in Article 5. EFSA shall deliver an opinion on the safety for the target animals, the user and the efficacy of the product Allura Red AC, when used under the conditions described in Table1.

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Commission Directive 2008/128/EC of 22 December 2008 laying down specific purity criteria concerning colours for use in foodstuffs. OJ L 6, 10.1.2009, p.20.

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Table 1:
Additive

Description and conditions of use of the additive as proposed by the applicant
Allura Red AC E 129 2 (Sensory) A (Colourants)

Registration number/EC No/No (if appropriate) Category(-ies) of additive Functional group(s) of additive

Description Composition, description Disodium 2-hydroxy-1-(2methoxy-5-methyl-4sulfonatophenylazo) naphthalene-6-sulfonate; Dark red powder or granules Chemical formula Purity criteria (if appropriate) Colour (Red); Spectrometry (504 nm); Assay (Minimum 85% total colouring matters calculated as sodium salt) Allura Red AC Not relevant Method of analysis (if appropriate) Annex II Methods of Analysis Relating to the Criteria of Purity of Food Additives Commission Directive 81/712/EEC

C18H14N2Na2O8S2

Trade name (if appropriate) Name of the holder of authorisation (if appropriate) Conditions of use Species or category of animal Cats and dogs Minimum content Maximum Age

Maximum content

mg/kg of complete feedingstuffs Not relevant Not relevant Not relevant

Withdrawal period (if appropriate) Not relevant

Other provisions and additional requirements for the labelling Specific conditions or restrictions for use (if appropriate) Specific conditions or restrictions for handling (if appropriate) Post-market monitoring (if appropriate) Specific conditions for use in complementary feedingstuffs (if appropriate)

Not relevant
Not relevant Not relevant Not relevant

Maximum Residue Limit (MRL) (if appropriate) Marker residue Not relevant Species or category of animal Not relevant Target tissue(s) or food products Not relevant Maximum content in tissues Not relevant

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Allura Red AC (E129) for cats and dogs

ASSESSMENT
The applicant asked for a re-evaluation of the use of Allura Red AC (E 129) in feedingstuffs for cats and dogs. Since the additive is only applied for non food-producing animals, the assessment of safety is limited to the target species and the user. 1. Introduction

Allura Red AC is included in the European Union Register of Feed Additives pursuant to Regulation (EC) No 1831/2003. It is authorised without a time limit in application of Article 9t (b) of Council Directive 70/524/EEC11 concerning additives in feedstuffs (2004/C 50/01) for its use in cats and dogs as a colourant additive (colouring agents authorised for colouring foodstuffs by Community rules). The additive is also authorised for all species or categories of animals, with the exception of cats and dogs, for animal feedstuffs only in products processed from (i) waste products of foodstuffs; and (ii) other base substances, with the exception of cereals and manioc flour, denatured by means of these agents or coloured during technical preparation to ensure the necessary identification during manufacture. No maximum levels for Allura Red AC in feeds are established in the EU. Allura Red AC, a synthetic food colouring substance, is an approved food colourant in the EU, and it is listed in Annex I of Directive 94/36/EC12 of 30 June 1994 for a limited number of foodstuffs with a maximum allowed usage level of 25–500 mg/kg food for various foodstuffs. Allura Red AC is also permitted in alcoholic beverages at levels up to 200 mg/l and non-alcoholic beverages at levels up to 100 mg/L. Allura Red AC has previously been evaluated by JECFA in 1974 (JECFA, 1974), 1980 (JECFA, 198013 and 1981 (JECFA, 1981)14 and the SCF in 1975 (EC, 1975), 1984 (EC, 1984)15 and 1989 (EC, 1989).16 Allura Red AC was evaluated in 2000 by the National Toxicological Program (NTP, 2000).17 In 2002, the Nordic Working Group on Food Toxicology and Risk Assessment (NNT, 2002)18 reviewed the current status and safety data on all food additives permitted in the EU, including Allura Red AC. The EFSA Panel on Food Additives and Nutrient Sources Added to Food (ANS) issued a scientific opinion on the re-evaluation of Allura Red AC as a food additive (EFSA, 2009). In 2010 the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) issued a scientific opinion on the appropriateness of the food azo-colours (including Allura Red AC) for inclusion in the list of food ingredients set up in Annex IIIa of Directive 2000/13/EC (EFSA, 2010). 2. Characterisation

The additive under application, Allura Red AC (E 129, synonymous CI Food Red 17, Food Red No. 40, FD&C Red No. 40), is identical to the active substance. Allura Red AC is a is a monoazo class of colourant, comprising primarily disodium 2-hydroxy-1-(2methoxy-5-methyl-4-sulphonato-phenylazo) naphthalene-6-sulphonate (C18H14N2Na2O8S2; CAS (Chemical Abstracts Service) number 25956-17-6, molecular weight 496.42) and subsidiary colouring matters, together with sodium chloride and/or sodium sulphate as the principal uncoloured components. Although typically described as the disodium salt, the calcium and potassium salts are also permitted. The structural formula of Allura Red AC is given in Figure 1. Allura Red AC is a water soluble dark red powder or granules.

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OJ C 50, 25.2.2004, p.1. OJ L 237, 10.09.1994, p. 13. 13 Technical Dossier/Section III/Annex 25. 14 Technical Dossier/Section III/Annex 26. 15 Technical Dossier/Section III/Annex 42. 16 Technical Dossier/Section III/Annex 43. 17 Technical Dossier/Section III/Annex 34. 18 Technical Dossier/Section III/Annex 33.

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Allura Red AC (E129) for cats and dogs

Figure 1

Structural formula of Allura Red AC

The specifications for Allura Red AC when used as a feed additive are identical to those for Allura Red AC when used as a food additive laid down in Commission Directive 2008/128/EC,19 establishing thresholds for colouring matters (a minimum content of 85 %, calculated as the sodium salt), water insoluble matter, subsidiary colouring matter, arsenic, lead, mercury, cadmium, heavy metals (expressed as Pb), organic compounds other than colouring matters (6-hydroxy-2-naphthalene sulfonic acid, sodium salt; 4-amino-5-methoxy-2-methylbenezene sulfonic and 6,6-oxybis (2-naphthalene sulfonic acid) disodium salt), unsulfonated primary aromatic amines and ether extractable matter). Analysis of five production batches20 confirmed compliance with these thresholds (89.3–90 % colouring matter). Control measures are in place. 2.1. Particle size

The additive is produced in two different forms, an ultra-fine powder for application as powder and a granular product for liquid application. The particle size distribution was determined by laser diffraction/scattering analysis in three batches from three manufacturers.21 In the ultra-fine powder the volume based percentage of particles < 1 m diameter was 100 %. The figures for the two granulated products were between 2 % and 10 % < 10 m, and between 45 % and 85 % < 50 m. Data for dusting potential were not available. 2.2. Manufacturing process

Allura Red AC is manufactured from 4-amino-5-methoxy-2-methylbenezene sulphonic acid, which is diazotised with hydrochloric acid and sodium nitrite then coupled to 6-hydroxynaphthalene-2sulphonic acid. 2.3. Stability and homogeneity

No data were submitted. The applicant reported a shelf-life of 4–6 years for Allura Red AC stored in a dry, cool and ventilated place, based on its own experience from the use of the product in food, cosmetics and other applications. Moreover, the applicant made reference to a control review22 conducted by the United States Office of Cosmetics and Colours (OCAC) of its procedures for the Colour Certification Program. A total of 175 sample batches were analysed (certified between 1963 and 1992). Of the samples analysed specifically, a few exhibited typical signs of moisture absorption and high volatile matter, accompanied by low percentage total colour. The applicant noted that the conditions of use for Allura Red AC in a range of foods are well established. Any substance that interacts with or alters conjugated unsaturated bonds of the molecule will affect the colour. Allura Red AC will generally be unstable in the presence of oxidising or reducing agents (e.g. sugars and acids).
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OJ L 6, 10.1.2009, p.20. Technical dossier/Section II/Annex 1. 21 Supplementary information October 2011. 22 Technical dossier/Section II/Annex 2.

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No data on the potential of the additive to homogeneously distribute in different feedingstuffs have been submitted. The applicant noted that, owing to its solubility, Allura Red AC will generally be distributed in the aqueous medium of the feedingstuff, independent of its route of application (either added directly as a solid to the feedingstuffs in the presence of water or by addition of an aqueous solution). 2.4. Conditions of use

Allura Red AC is intended to be used in complete and complementary feedingstuffs for cats and dogs without a maximum content. The applicant noted that the quantity required is dependent on the properties of the feedingstuff, but in general is not likely to exceed 500 mg/kg complete feedingstuffs. The additive may be added to feed directly or via an intermediary step by dissolving it in water but the additive is not intended to be added to water for drinking. 2.5. Evaluation of the analytical methods by the European Union Reference Laboratory (EURL)

EFSA has verified the EURL report as it relates to the methods used for the control of the active substance in the feed additive. The executive summary of the EURL report can be found in the Appendix. 3. Safety

The EFSA ANS Panel evaluated the toxicological data on Allura Red AC and concluded that the present data set does not give reason to revise the acceptable daily intake (ADI) of 7 mg/kg body weight (EFSA, 2009) already set by the JECFA (JECFA, 1980; JECFA, 1981) and the SCF (EC, 1984; EC, 1989). The ADI had been set on the basis of the results of two studies: a no observable adverse effect level (NOAEL) of 695 mg/kg body weight per day for growth suppression in pups of rats given up to 2595 mg/kg body weight per day in a reproduction toxicity study and a NOAEL in a developmental toxicity study in rats of 701 mg/kg body weight per day for males (and 901 mg/kg body weight per day for females) which was the highest dose given. 3.1. 3.1.1. Safety for the target animals Genotoxicity studies including mutagenicity

3.1.1.1. In vitro tests Allura Red AC was tested in several bacterial reverse mutation studies in Salmonella Typhimurium (EFSA, 2009). Although the studies were performed before the establishment of the current Organization for Economic Cooperation and Development (OECD) guidelines, the substance was tested in all the strains recommended by OECD guidelines. Allura Red AC gave negative results with and without metabolic activation. Allura Red AC also gave negative results in a modified protocol of the Ames test designated to detect mutagenic compounds that may result from azo-reduction of Allura Red AC (Prival et al., 1988). Negative results were reported also in a gene mutation assay with Saccharomyces cerevisiae23 (EFSA, 2009).

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These genotoxicity tests are obsolete and the relevant guidelines are in the list of those submitted to OECD Council for deletion.

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3.1.1.2. In vivo tests Allura Red AC was tested with negative results in the mouse heritable translocation test and in the recessive lethal test in Drosophilia melanogaster.24 Allura Red AC was tested for its ability to induce DNA damage in an in vivo comet assay in male mice and pregnant mice (Tsuda et al., 2001). Groups of male mice received by gavage doses of 0, 1, 10, 100, 1000 and 2000 mg Allura Red AC per kilogram body weight. Three hours after administration, Allura Red AC induced significant increases in migration of nuclear DNA in the glandular stomach at doses of 100 mg/kg body weight and higher. In the colon, significant differences between the treatment group and controls were observed at doses from 10 mg/kg body weight upwards; the effect was slightly dose related and reached a plateau at doses higher than 100 mg/kg body weight. In the lung, a slight increase in nuclear DNA migration was observed only at a dose of 1000 mg/kg body weight. In the group receiving the highest dose (2000 mg/kg body weight), nuclear DNA migration was also determined at 6 and 24 hours after administration and found to be significantly increased. A modest but significant effect was also observed in the colon of pregnant mice 3 hours after treatment with a dose of 2000 mg Allura Red AC/kg body weight. Necropsy and histopathological examination revealed no treatment-related effect on the colon and glandular stomach. The authors discussed that (i) the effect observed was not likely to be due to general cytotoxicity; and (ii) the DNA damage induced by Allura Red AC may have been due to adduct formation. They concluded that a more extensive safety assessment is needed because Allura Red AC induced DNA damage mainly in the colon at 10 mg/kg body weight, a dose close to the ADI. Another in vivo comet study was conducted in male mice and male rats in order to examine potential differences in response between those two species (Shimada et al., 2010). In mice Allura Red AC was confirmed to induce DNA damage in the colon 3 hours after oral administration (gavage) of 10 mg/kg body weight. In contrast, no significant effect was seen in rats receiving by gavage 10 mg/kg body weight after 3 hours and 100 and 1 000 mg/kg body weight after 6, 12 and 24 hours. There was no species difference in transit time through the gastrointestinal tract. 3.1.2. Conclusions from the genotoxicity tests

Allura Red AC was negative in bacterial reverse mutation assays. No other validated in vitro tests were provided. Allura Red AC was positive in two in vivo comet assays in mice (stomach and colon), but these findings were not seen in rats. On the basis of the experimental results, it is not possible to exclude a genotoxic potential of Allura Red AC. 3.1.3. Long-term studies

The ANS Panel re-evaluated long-term carcinogenicity studies on Allura Red AC (EFSA, 2009), which are summarised below: “Several long term carcinogenicity studies at dose levels up to 2595 mg/kg bw/day in male rats (Olson and Voelker, 1970) and 2829 mg/kg bw/day in female rats (Serota et al., 1977b; Borzelleca et al., 1989), and in mice at dose levels up to 7422 mg/kg bw/day in males and 8304 mg/kg bw/day in females (Serota et al., 1977a; Borzelleca et al., 1991; Reno et al., 1978), revealed no evidence of carcinogenicity. This included the absence of neoplasms in the stomach and the large intestine, shown to be the most sensitive organs in the in vivo Comet assay in mice. Allura Red AC induced significant dose-related DNA damage in mice in the glandular stomach at doses of 100 mg/kg bw and higher, and in the colon at doses of 10 mg/kg bw and higher. However, carcinogenicity in these tissues was not observed at dose levels several times higher up to 7422 and 8304 mg/kg bw/day for male and female mice, respectively (Serota et al., 1977a; Borzelleca et al., 1991; Reno et al., 1978). Therefore the Panel concluded that the effects on

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These genotoxicity tests are obsolete and the relevant guidelines are in the list of those submitted to the OECD Council for deletion.

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nuclear DNA migration observed in the mouse in vivo Comet assay are not expected to result in carcinogenicity.” All chronic studies available for Allura Red AC were negative for carcinogenicity. The EFSA Scientific Committee (EFSA, 2011) stated that “A decision on whether negative carcinogenicity data can over-rule positive in vitro genotoxicity test results should be taken on a case-by-case basis.” It further noted that “It is doubtful, though, whether this should also be the case for positive in vivo genotoxicity test results. Clear evidence of genotoxicity in somatic cells in vivo should be considered an adverse effect per se, since genotoxicity is also implicated in degenerative diseases other than cancer.” The absence of colon cancer in the mouse carcinogenicity studies is unsurprising, as the mouse is not a good model for detecting colon carcinogens. In a review of bioassays of 299 chemicals that were carcinogenic in mice, none caused cancer in the colon, although it showed that 4 % of 354 chemicals evaluated as carcinogenic in rats have the large intestine as the target organ (Gold et al., 1991). The sensitivity to carcinogenicity of the mouse colon seemed to be very low compared with the rat colon. 3.1.4. Conclusion

The FEEDAP Panel considered that (i) genotoxicity of Allura Red AC cannot be excluded; (ii) the mouse carcinogenicity study may not be appropriate for investigating the risk of colon cancer that might result from local DNA damage; and (iii) Allura Red AC is proposed for lifetime use in cats and dogs, leading to a much higher exposure in target animals than in humans. The FEEDAP Panel concluded that the available data are insufficient to demonstrate the safety of Allura Red AC for cats and dogs. 3.2. Safety for the user

With regard to potential allergenicity, the ANS Panel (EFSA, 2009) concluded that: “while some sensitivity reactions after Allura Red AC intake (urticaria, rhinitis and asthma) have been reported, mostly when Allura red AC is taken within mixtures of other synthetic colours, no conclusion on the induction of sensitivity by Allura Red AC could be drawn from the limited scientific evidence available. The Panel also notes that sensitive individuals may react at dose levels within the ADI.” The applicant performed on request a literature search concerning the skin and eye irritancy, skin sensitisation and inhalation toxicity of Allura Red AC in the following databases: MEDLINE, ToxFile, AGRICOLA, AGRIS, BIOSIS Toxline, FOODLINE (Science), Food Sci.&Tech.Abs, Biosis Previews, NTIS and EMBASE, covering a period from 1950 to August 2011. No studies were found. The ultra-fine powder and the granulated form of Allura Red AC have large proportions of particles of respirable size. No information was available on dusting potential. In the absence of any information, the substance should be considered to be potentially harmful as a result of skin, eye, or inhalation exposure. 4. Efficacy

Where the function requested for feed is the same as that used in food, no further demonstration of efficacy might be necessary (Regulation (EC) No 429/200825). However, considering the wide variety of feedingstuffs used in complete and complementary feed for cats and dogs and the uncertainty of which concentration of Allura Red AC would result in a visible effect, a demonstration of dose–effect in a typical complementary feedingstuffs was requested.
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Commission Regulation (EC) No 429/2008 of 25 April 2008 on detailed rules for the implementation of Regulation (EC) No 429/2008 of the European Parliament and of the Council as regards the preparation and presentation of applications and the assessment and authorisation of feed additives. OJ L 133, 22.5.2008, p.1.

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Samples of standard biscuits were prepared containing wholemeal flour, milk powder and vegetable oil. Allura Red AC was added at 0, 50 and 500 mg/kg feed. The colour of the samples was measured by reflectance spectrophotometry. By the addition of Allura Red AC the “a” value increased from 7.4 (blank sample), to 23.3 (50 mg Allura Red AC) and 35.7 (500 mg Allura Red AC). The “L” value decreased accordingly (from 61.0 to 48.3 and 39.6, respectively) and the “b” value from 22.4 to 20.5 and 18.5, respectively. 26 The data demonstrated that Allura Red AC is effective in colouring a typical feed for dogs at a minimum dose of 50 mg/kg.

CONCLUSIONS
The FEEDAP Panel considered that (i) genotoxicity of Allura Red AC cannot be excluded; (ii) the mouse carcinogenicity study may not be appropriate for investigating the risk of colon cancer that might result from local DNA damage; and (iii) Allura Red AC is proposed for lifetime use in cats and dogs, leading to a much higher exposure in target animals than in humans. The FEEDAP Panel concluded that the available data are insufficient to demonstrate the safety of Allura Red AC for cats and dogs. In the absence of any information, the substance should be considered to be potentially harmful as a result of skin, eye, or inhalation exposure. Allura Red AC is considered efficacious in adding colour to feed for cats and dogs at a minimum dose of 50 mg/kg. RECOMMENDATION The specification for Allura Red AC for feed use should follow those set for food use (Commission Regulation (EU) No 231/2012).27

DOCUMENTATION PROVIDED TO EFSA
1. 2. 3. 4. 5. Allura Red AC. November 2010. Submitted by Feed Additives Synthetic Colours Group. Allura Red AC. Supplementary information. October 2011. Submitted by Feed Additives Synthetic Colours Group. Allura Red AC. Supplementary information. January 2012. Submitted by Feed Additives Synthetic Colours Group. Evaluation report of the European Union Reference Laboratory for Feed Additives on the methods(s) of analysis for Allura Red AC. Comments from Member States received through ScienceNet.

REFERENCES
EC (European Commission), 1975, online. Reports of the Scientific Committee for Food (1st Series), opinion expressed on 31 December 1975. Available from http://ec.europa.eu/food/fs/sc/scf/reports/scf_reports_01.pdf

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Colour quantification standardised by the Commission International de l’Eclairage. L (lightness, black to white reflectance, 1–100), a (red=positive, green=negative), b (yellow, blue). 27 Commission Regulation (EU) No 231/2012 of 9 March laying down specification for food additives listed in Annex II and III to Regulation 1333/2008 of the European Parliament and of the Council . OJ L 83, 22.3.2012, p. 1.

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EC (European Commission), 1984, online. Reports of the Scientific Committee for Food (14th Series), opinion expressed on 7 July 1983. Available from http://ec.europa.eu/food/fs/sc/scf/reports/scf_reports_14.pdf EC (European Commission), 1989. Reports of the Scientific Committee for Food (21st Series), opinion expressed on 10 December 1987. EFSA (European Food Safety Authority), 2009. Scientific opinion on the reevaluation of Allura Red AC (E 129) as a food additive on request from the European Commission. EFSA Journal 7(11): 1327. EFSA (European Food Safety Authority), 2010. EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA); Scientific opinion on the appropriateness of the food azo-colours Tartrazine (E 102), Sunset Yellow FCF (E 110), Allura Red AC (E 122), Amaranth (E 123), Ponceau 4R (E 124), Allura Red AC (E 129), Brilliant Black BN (E 151), Brown FK (E 154), Brown HT (E 155) and Litholrubine BK (E 180) for inclusion in the list of food ingredients set up in Annex IIIa of Directive 2000/13/EC. EFSA Journal 8(10): 1778. EFSA (European Food Safety Authority), 2011. EFSA Scientific Committee. Scientific opinion on genotoxicity testing strategies applicable to food and feed safety assessment. EFSA Journal 9(9): 2379. Gold LS, Slone TH, Manley NB, Bernstein L, 1991. Target organs in chronic bioassays of 533 chemical carcinogens. Environmental Heath Perspective 93, 233–246. JECFA (Joint FAO/WHO Expert Committee on Food Additives), 1974. Report of the Joint FAO/WHO Expert Committee on Food Additives, WHO Technical Report Series, No. 557. JECFA (Joint FAO/WHO Expert Committee on Food Additives), 1980. 24th Report. Toxicological evaluation of certain food additives. WHO Food Additive Series, No. 15. JECFA (Joint FAO/WHO Expert Committee on Food Additives), 1981. 25th Report. Evaluation of certain food additives. WHO Technical Report Series, No. 669. NNT (Nordic Working Group on Food Toxicology and Risk Assessment), 2002. Food additives in Europe 2000 – Status of safety assessments of food additives presently permitted in the EU. NTP (National Toxicology Program), 2000. Allura red. Available from NTP website: http://ntp.niehs.nih.gov/index.cfm?objectid=E87EF85F-BDB5–82F8-FDED9AB34EFB2FAD Prival MJ, Davis VM, Peiperl MD and Bell SJ, 1988. Evaluation of azo dyes for mutagenicity and inhibition of mutagenesis by methods using Salmonella typhimurium. Mutation Research 206, 247–259. Shimada C, Kano K, Sasaki, YF, Sato I, Tsuda S, 2010. Differential colon damage induced by azo food additives between rats and mice. Journal of Toxicological Sciences 35, 547–554. Tsuda S, Murakami M, Matsusaka N, Kano K, Taniguchi K and Sasaki YF, 2001. DNA damage induced by red food dyes orally administered to pregnant and male mice. Toxicological Sciences 61, 92–99.

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Allura Red AC (E129) for cats and dogs

APPENDIX
Executive summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of Analysis for Allura Red AC28 In the current application authorisation is sought under articles 4(1) and 10(2) for Allura Red AC under the “sensory additives”, functional group 2(a) “colourants”, according to the classification system of Annex I of Regulation (EC) No 1831/2003. Authorisation is sought for the use of the feed additive for cats and dogs. Allura Red AC is a synthesized dark red powder or granules, soluble in water, consisting of a minimum of 85 % total colouring matters calculated as the sodium salt. Allura Red AC is intended to be incorporated directly in feedingstuff as a solution in water (either added directly as a solid to the feedingstuffs in the presence of water or by addition of an aqueous solution), with no recommended minimum or maximum levels. For the determination of Allura Red AC in the feed additive, the applicant proposed the internationally recognised FAO JECFA monograph for food additives. Identification and quantification of Allura Red AC in the feed additive are based on spectrophotometry at 504 nm in aqueous solution at pH 7, as recommended by Commission Directive 2008/128/EC laying down specific purity criteria concerning colours for use in foodstuffs. Even though no performance characteristics are provided, the EURL recommends for official control the JECFA monograph based on spectrophotometry for the quantification of the Allura Red AC in the feed additive. The applicant did not provide any experimental method or data for the determination of Allura Red AC in premixtures, feedingstuffs and water. Therefore the EURL cannot evaluate nor recommend any method for official control to determine Allura Red AC in premixtures, feedingstuffs and water. Further testing or validation of the methods to be performed through the Consortium of National Reference Laboratories as specified by Article 10 (Commission Regulation (EC) No 378/2005) is not considered necessary.

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The full report is available on the EURL website: http://irmm.jrc.ec.europa.eu/SiteCollectionDocuments/FinRep-FAD2010-0347.pdf

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