Scientific Opinion on the safety and efficacy of erythrosine in feed for cats and dogs, ornamental fish and reptiles 1

SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of erythrosine in feed for cats and dogs, ornamental fish and reptiles 1 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3 ABSTRACT European Food Safety Authority (EFSA), Parma, Italy Erythrosine (E 127), an approved colourant in food, is intended to be used in feedingstuffs for cats, dogs, ornamental fish and reptiles without quantitative limitation. Since the additive is only applied for non foodproducing animals, the assessment of safety was limited to the target species and the user. The FEEDAP Panel concludes that the maximum use level indicated by the applicant (500 mg/kg complete feed) is not safe for the target species cats and ornamental fish. Based on data from a two-year study in dogs, with a limited range of endpoints, and applying a safety factor of 10, 1000 mg erythrosine/kg complete feed could be derived as the maximum safe concentration. No conclusion could be drawn on the safety of erythrosine for reptiles. However, since erythrosine contains 56.5 % iodine, compliance with the maximum iodine content established in the EU for feed of different animal species would limit the maximum erythrosine content to 35 mg/kg complete feed for ornamental fish and 18 mg/kg complete feed for the other target species. Erythrosine was not irritant to skin or eyes in rabbits. It was not a skin sensitiser in guinea pigs and mice and was also negative in human skin patch testing. However, dermatological reactions, including photosensitivity, erythroderma and desquamation, have been attributed to erythrosine. Since 90 % of the particles have a diameter < 10 m, an exposure of the lower respiratory tract was considered a hazard. No inhalation toxicity studies were available. A quantitative assessment of the efficacy of erythrosine in adding colour to feedingstuffs appeared necessary considering the variety of feed materials used in the relevant complementary and complete feedingstuffs and the very limited food use. An assessment of efficacy with respect to the variables mentioned was not possible due to the absence of data. European Food Safety Authority, 2011 KEY WORDS Erythrosine, E127, colourant, cats, dogs, ornamental fish, reptiles, safety 1 On request from the European Commission, Question No EFSA-Q-2010-01527, adopted on 16 November 2011. 2 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 3 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 erythrosine in feed for cats and dogs, ornamental fish and reptiles. EFSA Journal 2011;9(12):2447. [13 pp.]. doi:10.2903/j.efsa.2011.2447. Available online: www.efsa.europa.eu/efsajournal European Food Safety Authority, 2011

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 erythrosine (E 127) in feed for cats and dogs, ornamental fish and reptiles. Erythrosine is already approved for feed and food use. Erythrosine is intended to be used as a colourant in feedingstuffs for cats, dogs, ornamental fish and reptiles without quantitative limitations. Since the additive is only applied for non food-producing animals, the assessment of safety was limited to the target species and the user. The FEEDAP Panel concluded that the maximum use level indicated by the applicant (500 mg/kg complete feed) is not safe for the target species cats and ornamental fish. Based on data from a twoyear study in dogs, with a limited range of endpoints, and applying a safety factor of 10, 1000 mg erythrosine/kg complete feed could be derived as the maximum safe concentration. No conclusion could be drawn on the safety of erythrosine for reptiles. However, since erythrosine contains 56.5 % iodine, compliance with the maximum iodine content established in the EU for feed of different animal species would limit the maximum erythrosine content to 35 mg/kg complete feed for ornamental fish and 18 mg/kg complete feed for the other target species. Erythrosine was not irritant to skin or eyes in rabbits. It was not a skin sensitiser in guinea pigs and mice and was also negative in human skin patch testing. However, dermatological reactions, including photosensitivity, erythroderma and desquamation, have been attributed to erythrosine. Since 90 % of the particles have a diameter < 10 m, an exposure of the lower respiratory tract was considered a hazard. No inhalation toxicity studies were available. A quantitative assessment of the efficacy of erythrosine in adding colour to feedingstuffs appeared necessary considering the variety of feed materials used in the relevant complementary and complete feedingstuffs and the very limited food use. An assessment of efficacy with respect to the variables mentioned was not possible due to the absence of data. 2

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. Characterisation of additive... 7 2.2. Stability and homogeneity... 8 2.2.1. Shelf life... 8 2.2.2. Stability in premixture and feedingstuffs... 8 2.2.3. Homogeneity... 9 2.3. Conditions of use... 9 2.4. Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) 9 3. Safety... 9 3.1. Safety for the target species... 9 3.1.1. Conclusion on the safety for the target species... 10 3.2. Safety for the user... 10 4. Efficacy... 10 Conclusions... 10 Documentation provided to EFSA... 11 References... 11 Appendix... 13 3

BACKGROUND Regulation (EC) No 1831/2003 4 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 addition, 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/EEC 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 a time limit or pursuant to Directive 82/471/EEC. The European Commission received a request from the company Roha UK Ltd 5 for re-evaluation of the product erythrosine (E127), when used as a feed additive for cats and dogs, ornamental fish and the authorisation for use in reptiles (category: sensory additive; functional group: 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. 6 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 1 July 2011. Erythrosine (E127) 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 7 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. 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 ornamental fish as colourant additive. No maximum levels of erythrosine (E127) in feeds are established in the EU. The applicant asks for the re-evaluation of the use of the additive for cats and dogs, ornamental fish and reptiles. Erythrosine (E 127) has a long history of use as an additive in food and pharmaceuticals. Erythrosine (E127) is an approved food colourant in the EU and it is listed in Annex I of Directive 94/36/EC 8 of 30 June 1994. It is exclusively authorised for use in cocktail and candied cherries and bigarreaux cherries. The specific purity criteria concerning the use of erythrosine (E127) in foodstuffs are included in Commission Directive 2008/128/EC. 9 Erythrosine (E127) has previously been evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1986 and 1990 (JECFA, 1986, 1990) and the EU Scientific Committee for Food (SCF) in 1989 (EC, 1989). The Scientific Committee on Consumer Safety (SCCS) published an 4 OJ L 268, 18.10.2003, p. 29. 5 Roha UK Ltd. Unit 6A, Lodgeway, Severn Bridge Industrial Estate, NP26 5PT, Calidcot, UK. 6 EFSA Dossier reference: FAD-2010-0382. 7 OJ C 50, 25.2.2004, p. 1. 8 OJ L 237, 10.09.1994, p. 13. 9 OJ L 6, 10.1.2009, p. 20. 4

opinion in 2010 (EC, 2010). EFSA Panel on food additives and nutrient sources added to food (ANS) issued a scientific opinion on the re-evaluation of erythrosine (E127) as a food additive in 2011 (EFSA, 2011). 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, consumer, user and the environment and the efficacy of the product erythrosine (E127), when used under the conditions described in Table1. 5

Table 1: Description and conditions of use of the additive as proposed by the applicant Additive Registration number/ec No/No (if appropriate) Category(-ies) of additive Functional group(s) of additive Erythrosine E 127 2 (Sensory) A (Colourants) Composition, description Disodium 2-(2,4,5,7-tetraiodo-3- oxido-6-oxoxanthene-9-yl) benzoate monohydrate; red powder or granules Chemical formula C 20 H 6 I 4 Na 2 O 5 H 2 O Description Purity criteria (if appropriate) Colour (red); Spectrometry (526 nm); Assay (Minimum 87% total colouring matters calculated as anhydrous sodium salt) Method of analysis (if appropriate) Annex II Methods of Analysis Relating to the Criteria of Purity of Food Additives Commission Directive 81/712/EEC Trade name (if appropriate) Name of the holder of authorisation (if appropriate) Erythrosine Not relevant Conditions of use Species or category of animal Cats and dogs, ornamental fish and reptiles Maximum Age Minimum content Maximum content Withdrawal period mg/kg of complete feedingstuffs (if appropriate) Not relevant Not relevant Not relevant 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 Marker residue Maximum Residue Limit (MRL) (if appropriate) Species or category of animal Target tissue(s) or food products Maximum content in tissues Not relevant Not relevant Not relevant Not relevant 6

ASSESSMENT 1. Introduction The applicant asks for the re-evaluation of the use of erythrosine (E 127) in feedingstuffs for cats, dogs and ornamental fish and for a new use in feedingstuffs for reptiles. Since the additive is only applied for non food-producing animals, the assessment of safety is limited to the target species and the user. Erythrosine is included in the European Union Register of Feed Additives pursuant to Regulation (EC) No 1831/2003. 10 It is authorised without a time limit in application of Article 9t (b) of Council Directive 70/524/EEC 11 concerning additives (2004/C 50/01) for its use in feedingstuffs for cats, dogs and ornamental fish as a colourant additive. The additive is further authorised for all species or categories of animals with the exception of cats and dogs in 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 erythrosine in feeds and no MRLs are established in the EU. Erythrosine is an approved food colourant in the EU and it is listed in Annex I of Directive 94/36/EC. 12 It is authorised for use in cocktail and candied cherries up to 200 mg/kg, and bigarreaux cherries up to 150 mg/kg. Erythrosine is permitted to be used as a colour in cosmetics, 13 pharmaceuticals 14 and for use in pesticide formulation 15 in the EU. Erythrosine has previously been evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1986 and 1990 (JECFA, 1986, 1990) and the EU Scientific Committee for Food (SCF) in 1989 (EC, 1989). The Scientific Committee on Consumer Safety (SCCS) published an opinion in 2010 on its use in toothpaste products (EC, 2010). 16 EFSA Panel on food additives and nutrient sources added to food (ANS) has issued a scientific opinion on the re-evaluation of erythrosine as a food additive (EFSA, 2011). 2. Characterisation 2.1. Characterisation of additive The additive under application, erythrosine (E 127, synonymous: CI Food Red 4, Acid Red 51, FD&C Red No 3), is identical to the active substance. Erythrosine contains mainly disodium 2-(2,4,5,7-tetraiodo-3-oxido-oxox-anthen-9-yl) benzoate monohydrate (chemical formula C 20 H 6 I 4 Na 2 O 5 H 2 O; CAS number 16423-68-0, molecular weight 897.88) and subsidiary colouring matter together with water, sodium chloride and/or sodium sulphate as the principal uncoloured components. While usually described as the sodium salt, the calcium and the potassium salts are also permitted. The structural formula of erythrosine is given in Figure 1. 10 OJ L 268, 18.10.2003, p. 29. 11 OJ C 50, 25.2.2004, p. 1. 12 OJ L 237, 10.09.1994, p. 13. 13 Technical dossier/section II/Annex 9, 10, 11. 14 Technicl dossier/section II/Annex 4, 5, 7. 15 Technical dossier/section II/Annex 12. 16 Technical dossier/section III/Annex 6. 7

Figure 1: Structural formula of erythrosine Erythrosine is a water soluble red powder or granules with the following characteristic of the sodium salt: solubility in water 70 g/l at 20 C, partition coefficient 4.95 at 25 C and ph 7 to 9 in aqueous solution. According to the applicant, the specifications for erythrosine when used as a feed additive are identical to those for erythrosine when used as a food additive laid down in Commission Directive 2008/128/EC. 17 This Directive established thresholds for erythrosine (not less than 87 % total colouring matter, calculated as the anhydrous sodium salt), sodium iodide, water insoluble matter, subsidiary colouring matter (except fluorescin), triiodoresorcinol, 2-(2,4-dihydroxy-3,5-diodobenzoyl) benzoic acid, ether extractable matter and for heavy metals and arsenic. The analysis of five production batches confirmed compliance with those thresholds 18 and control measures are in places. Erythrosine is prepared by iodination of 9-(O-carboxyphenyl)-6-hydroxy-3H-xanthen-3-one, disodium salt. Depending on the intended use in feedingstuffs as a solid form or aqueous solution, fine powders or coarse granules are produced. The particles size distribution determined by laser diffraction/scattering analysis from one batch of the fine powder was provided. 19 The additive had 100 % particles (v/v) with a diameter < 50 m, 90 % < 10 m and 13 % < 1 m. 2.2. Stability and homogeneity 2.2.1. Shelf life No data were submitted. The applicant reported the establishment of a shelf life of six years for erythrosine stored at room temperature in a closed container based on experiments supporting its use in food and cosmetics. 20 2.2.2. Stability in premixture and feedingstuffs No data on stability in premixture or feedingstuffs has been submitted. The applicant stated that erythrosine will generally be unstable in the presence of oxidising or reducing agents, sugars, acids, salts. Thermal degradation studies over the temperature range 20 350 C indicate that free iodide is only released from erythrosine at temperatures exceeding 200 C (Barbano and Deilavalle, 1984), which will not normally occur in feed processing. 17 OJ L 6, 10.1.2009, p. 20. 18 Technical dossier/section II/Annex 1. 19 Supplemetary information/september 2011. 20 Technical dossier/section II. 8

2.2.3. Homogeneity Erythrosine (E127) for cats and dogs, ornamental fish and reptiles No data and no demonstration that the additive has a potential to distribute homogenously in different feedingstuffs has been submitted. 2.3. Conditions of use Erythrosine is intended to be used in complete and complementary feedingstuffs for dogs, cats, ornamental fish and reptiles. The inclusion level may vary from about 5 mg/kg for sticks/treats to about 13 mg/kg for complete feed of dogs and cats. Maximum inclusion levels in feedingstuffs for reptiles and ornamental fish are stated to be up to 2000 and 7000 mg/kg, respectively. However, since those feedingstuffs are mostly complementary feeds, the applicant estimates that 500 mg/kg feed is typically not exceeded and proposes this level as the worst case exposure for all target animals. 21 2.4. 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 ANS Panel (EFSA, 2011) concluded that the present database on genotoxicity, sub-chronic, reproductive/developmental and long-term toxicity, does not provide a reason to revise the ADI of 0 0.1 mg/kg bw/day previously set by JECFA (JECFA, 1986, 1990) and the SCF (EC, 1989). The critical toxic effect was on thyroid hormone levels affected by the iodine from erythrosine. 3.1. Safety for the target species The only toxicity study performed in target animals was found in a publication from 1973. 22 Groups of three male and three female beagle dogs were fed diets containing 0, 0.5, 1.0 and 2.0 % erythrosine for two years. Considering the occurrence of chronic thyroiditis in one male and one female of the 2.0 % group, the FEEDAP Panel concludes that 1 % erythrosine in the diet could be regarded as the No Observed Adverse Effect Level (NOAEL). Considering individual variation and the small number of animals per group, a safety factor of 10 should be applied when deriving a safe level for complete feedingstuffs for dogs. Consequently, up to 1000 mg erythrosine/kg complete feedingstuffs is considered safe for dogs. Several chronic and sub-chronic toxicity studies in different species are described in the ANS opinion (EFSA, 2011). The rat appeared to be the most sensitive species. From the studies with rats, a NOAEL of about 0.25 % in the diet could be identified based on effects on thyroid function and related hormones. An estimation made for ornamental fish 23 identified about 240 mg/kg feedingstuffs (277 mg/kg dry matter) as the upper safe level. A comparable calculation for cats (default values: 3 kg body weight, 60 g feed dry matter daily intake) resulted in an upper safe level of 55 mg/kg complete feed (62.5 mg/kg dry matter). Since erythrosine contains 56.5 % iodine, the tolerance of the target animals to iodine should also be considered. Limited information on the metabolism of iodine in erythrosine indicated that 25 to 33 % of iodine from erythrosine was excreted within 48 hours as iodide in the urine of rats given 131 [I]- 21 Supplemetary information/september 2011. 22 Technical dossier/section III/Annex 1. 23 Based on a NOAEL from a rat study of 0.25 % erythrosine in feed corresponding to 125 mg/kg bw, applying a safety factor of 100 and using default values of 12 g of body weight for ornamental fish (goldfish, Carassius auratus) and 54 mg daily dry matter consumption (three-fold maintenance requirement of 18 mg/technical dossier/section III/Annex 4). 9

erythrosine orally (Vought et al., 1972). Considering that the upper safe level of dietary iodine for cats is 5.9 mg/kg (EFSA, 2005) and using bioavailability figures of iodine from erythrosine of 33 %, the upper safe level of erythrosine in feed for cats would be 18 mg erythrosine/kg feed. However, Regulation (EC) No 1459/2005 24 established a maximum content of 10 mg total iodine/kg complete feed for species or categories of animals other than equines, dairy cows and laying hens, and of 20 mg/kg for fish. Considering the maximum limits and the iodine content of erythrosine (56.5 % w/w), the maximum supplementation of erythrosine would be 35 mg/kg complete feed for ornamental fish and 18 mg/kg complete feed for the other target species when neglecting the background concentration of iodine and assuming that no other source of iodine is added. 3.1.1. Conclusion on the safety for the target species The FEEDAP Panel concludes that the maximum use level indicated by the applicant (500 mg/kg complete feed, see Section 2.3) is not safe for cats and ornamental fish. No conclusion can be drawn on the safety of erythrosine for reptiles. Very limited data indicate that 1000 mg erythrosine/kg complete feed may be safe for dogs (based on a toxicity study in dogs), that about 240 mg/kg is safe for ornamental fish (derived from toxicity studies in rats) and that about 18 mg erythrosine/kg is safe for cats (based on the iodine tolerance for cats). 3.2. Safety for the user Erythrosine was not irritant to skin or eyes in rabbits. It was not a skin sensitiser in guinea pigs and mice and was also negative in human skin patch testing. 25 A review of the Committee on Drugs of the American Academy of Pediatrics (AAP, 1997) contains among others, reports on adverse findings related to the use of erythrosine. Dermatological reactions, including photosensitivity, erythroderma and desquamation, have been attributed to erythrosine. Patients with the classic aspirin triad reaction (asthma, urticaria and rhinitis) or anaphylactoid reactions may also develop similar reactions from different dyes including erythrosine. Since 90 % of the particles have a diameter < 10 m, an exposure of the lower respiratory tract is considered a hazard. No inhalation toxicity studies were available. 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 429/2008). 26 Considering the wide range of use levels (5 to 7000 mg/kg feedingstuffs), the variety of feed materials and complete/complementary feedingstuffs to be treated with the additive compared to the very limited food use, and the range of maximum safe levels for the different target species (18 to 1000 mg/kg complete feed), the FEEDAP Panel is of the opinion that visual evidence of the effect of a certain dose of erythrosine in a certain feed material should be provided. The applicant considered those demonstrations as proprietary and consequently did not submit any requested data (photographic evidence of efficacy). Consequently, an assessment of efficacy with respect to the variables mentioned above was not possible. CONCLUSIONS The FEEDAP Panel concludes that the maximum use level indicated by the applicant (500 mg/kg complete feed) is not safe for the target species cats and ornamental fish. Based on data from a two- 24 OJ L 233, 9.9.2005, p. 8. 25 Technical dossier/section III/Annex 6. 26 OJ L 133, 22.5.2008, p. 1. 10

year study in dogs with a limited range of endpoints and applying a safety factor of 10, 1000 mg/kg complete feed could be derived as the maximum safe concentration. No conclusion can be drawn on the safety of erythrosine for reptiles. Erythrosine is not irritant to skin or eyes in rabbits. It is not a skin sensitiser in guinea pigs and mice and is also negative in human skin patch testing. However, dermatological reactions, including photosensitivity, erythroderma and desquamation, have been attributed to erythrosine. Since 90 % of the particles have a diameter < 10 m, an exposure of the lower respiratory tract is considered a hazard. No inhalation toxicity studies were available. A quantitative assessment of the efficacy of erythrosine in adding colour to feedingstuffs appeared necessary considering the variety of feed materials used in the relevant complementary and complete feedingstuffs and the very limited food use. An assessment of efficacy with respect to the variables mentioned is not possible due to the absence of data. REMARKS Considering the maximum content set by Regulation (EC) No 1459/2005 27 for iodine in feed and the iodine content of erythrosine, the maximum supplementation of erythrosine in accordance with that Regulation would be 35 mg/kg complete feed for ornamental fish and 18 mg/kg complete feed for the other target species. DOCUMENTATION PROVIDED TO EFSA 1. Erythrosine. November 2010. Submitted by Roha UK Ltd. 2. Erythrosine. Supplementary information. September 2011. Submitted by Roha UK Ltd. 3. Evaluation report of the European Union Reference Laboratory for Feed Additives on the methods(s) of analysis for Erythrosine. 4. Comments from Member States received through the ScienceNet. REFERENCES AAP (American Academy of Pediatrics), 1997. Inactive Ingredients in Pharmaceutical Products: Update (Subject Review). Pediatrics, 99, 268-278. Barbano DD and Deilavalle ME, 1984. Thermal degradation of FD&C Red No. 3 and release of free iodide. Journal of Food Protection, 47, 668-669. European Commission (EC), 1989, online. Reports of the Scientific Committee for Food (21st series), opinion expressed on 10 December 1987. Available from http://ec.europa.eu/food/fs/sc/scf/reports/scf_reports_21.pdf European Commission (EC), 2010, online. Scientific Committee on Consumer Safety. Opinion on CI 45430 (Erythrosine). Available from http://ec.europa.eu/health/scientific_committees/consumer_safety/docs/sccs_o_030.pdf EFSA (European Food Safety Authority), 2005. Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed on the request from the Commission on the use of iodine in feedingstuffs. The EFSA Journal, 168, 1-42. EFSA Panel on Food Additives and Nutrient Sources Added to Food (ANS), 2011. Scientific Opinion on the re-evaluation of Erythrosine (E 127) as a food additive. EFSA Journal, 9(1):1854. 27 OJ L 233, 9.9.2005, p.8. 11

JECFA (Joint FAO/WHO Expert Committee on Food Additives), 1986. Toxicological Evaluation of some food colours, enzymes, flavour enhancers, thickening agents, and certain food additives. WHO Food Additive Series 21. Erythrosine. JECFA (Joint FAO/WHO Expert Committee on Food Additives), 1990. Erythrosine. In: Evaluation of certain food additives and contaminants. WHO Technical Report Series, No. 806, 1991 and corrigenda/who Food Additive Series No. 28. Vought RL, Brown FA and Wolff J, 1972. Erythrosine: An adventitious source of iodide. Journal of Clinical Endocrinology & Metabolism, 34, 747-752. 12

APPENDIX Executive Summary of the Evaluation Report of the European Union Reference Laboratory for Feed Additives on the Method(s) of Analysis for Erythrosine 28 In the current application authorisation is sought under articles 4(1) and 10(2) for Erythrosine 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, ornamental fish, reptiles. Erythrosine is a synthesized red powder or granules, soluble in water, consisting of a minimum of 87 % total colouring matters calculated as anhydrous sodium salt. Erythrosine is intended to be incorporated directly in feedingstuffs 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 Erythrosine in the feed additive, the Applicant proposed the internationally recognised FAO JECFA monograph in food additives. Identification is based on spectrophotometry and Thin Layer Chromatography (TLC), while quantification of Erythrosine in the feed additive is based on spectrophotometry at 526 nm in aqueous solution, 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 Erythrosine in the feed additive. The Applicant did not provide any experimental method or data for the determination of Erythrosine in premixtures, feedingstuffs and water. Therefore the EURL cannot evaluate nor recommend any method for official control to determine Erythrosine 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. 28 The full report is available on the EURL website. http://irmm.jrc.ec.europa.eu/sitecollectiondocuments/finrep-fad- 2010-0382.pdf 13