astaxanthin dimethyldisuccinate

The EFSA Journal (2007) 574, 1-25

© European Food Safety Authority, 2007

Safety and efficacy of CAROPHYLL® Stay-Pink (astaxanthin dimethyldisuccinate) as feed additive for salmon and trout1

Scientific Opinion of the Panel on Additives and Products or Substances used in Animal Feed

(Question No EFSA-Q-2007-018)

Adopted on 17 October 2007 PANEL MEMBERS Georges Bories, Paul Brantom, Joaquim Brufau de Barberà, Andrew Chesson, Pier Sandro Cocconcelli, Bogdan Debski, Noël Dierick, Anders Franklin, Jürgen Gropp, Ingrid Halle, Christer Hogstrand, Joop de Knecht, Lubomir Leng, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Miklós Mézes, Carlo Nebbia, Walter Rambeck, Guido Rychen, Atte von Wright and Pieter Wester

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 a scientific opinion on the safety and efficacy of astaxanthin dimethyldisuccinate (CAROPHYLL® Stay-Pink) as a feed additive for salmon and trout.

CAROPHYLL® Stay-Pink (C®SP) is a feed additive, containing about 11 % astaxanthin dimethyldisuccinate (ATX-dimethyldisuccinate). The purpose of C®SP is to provide farmed Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) with a source of the carotenoid astaxanthin (ATX) which confers the characteristic pink colour to wild salmonids.

Orally administrated ATX-dimethyldisuccinate is hydrolysed and converted to free ATX in the intestine of fish, then absorbed, metabolised and distributed in the same manner as free ATX.

ATX-dimethyldisuccinate is efficacious in colouring the flesh of salmonids. This conclusion is based on studies comparing ATX-dimethyldisuccinate with free synthetic ATX, which produced similar results concerning flesh redness and ATX content in salmon and trout. Efficacy was demonstrated in the dose range of 20 to 100 mg ATX equivalents kg-1 feed, redness with tissue deposition reaching a plateau at about 60 mg ATX equivalents kg-1 feed.

A dietary level of 908 mg ATX equivalents kg-1 complete diet provided as ATX-dimethyldisuccinate from C®SP was well tolerated in rainbow trout for eight weeks. Although the short duration of the trial reduced the value of the study, taking into account former assessments of ATX, the FEEDAP Panel considers ATX-dimethyldisuccinate, at the highest authorised ATX level in feed, safe for the target species. 1 For citation purposes: Scientific Opinion of the Panel on Additives and Products or Substances used in Animal Feed

(FEEDAP) on a request from the European Commission on the safety and efficacy of CAROPHYLL® Stay-Pink (astaxanthin dimethyldisuccinate) as feed additive for salmon and trout. The EFSA Journal (2007) 574, 1-25

CAROPHYLL® Stay-Pink for salmon and trout


The data submitted in the dossier indicated particularly that the colourant was not genotoxic (ATX-dimethyldisuccinate), carcinogenic or teratogenic (ATX). Consequently, supplementation of fish feed with ATX at the highest approved level (100 mg kg-1 feed) is unlikely to represent an additional risk to the consumer. Given the nature of ATX-dimethyldisuccinate, its breakdown in the alimentary tract to ATX and its deposition as ATX in flesh, the FEEDAP Panel considers that no new or additional safety concerns from the use of ATX-dimethyldisuccinate, a substitutive product of other ATX sources, would originate for consumers.

No dermal or ocular risk for the users of C®SP is likely to occur under practical conditions. In the absence of an acute inhalation study, the acute inhalatory toxicity cannot be established. However, since the acute oral toxicity is very low (>2000 mg kg-1 bw) and particle size is >150 µm, systemic exposure through the respiratory tract is considered of limited risk.

The FEEDAP Panel does not expect that the use of ATX-dimethyldisuccinate will pose a risk to the environment.

The FEEDAP Panel recommends a number of modifications to the Register entry proposed by the applicant. In particular, it is recommended to include the content of ATX-dimethyldisuccinate and ATX equivalents of C®SP, a maximum content of dichloromethane and a reduction of the maximum TPPO content.

Key words: sensory additive, colourant, CAROPHYLL® Stay-Pink, red carotenoid, astaxanthin, astaxanthin dimethyldisuccinate, efficacy, toxicity, safety, salmon, trout, TPPO, dichloromethane



TABLE OF CONTENTS Panel Members .............................................................................................................................. 1 Summary........................................................................................................................................ 1 Table of Contents .......................................................................................................................... 3 Background ................................................................................................................................... 4 Terms of reference......................................................................................................................... 4 Acknowledgements ....................................................................................................................... 4 Assessment .................................................................................................................................... 7 1. Introduction ........................................................................................................................... 7 2. Characterisation of the product .............................................................................................7

2.1. Product identity..........................................................................................................................7 2.2. Purity..........................................................................................................................................8 2.3. Stability......................................................................................................................................8 2.4. Homogeneity..............................................................................................................................8 2.5. Evaluation of the analytical methods by the Community Reference Laboratory (CRL)...........9

3. Efficacy.................................................................................................................................. 9 3.1. Efficacy trials in Atlantic salmon (Salmo salar L.) ...................................................................9 3.2. Efficacy trials in trout (Oncorhynchus mykiss)........................................................................10 3.3. Other effects on the quality of fish flesh..................................................................................11 3.4. Conclusions on efficacy of astaxanthin from CAROPHYLL® Stay-Pink ...............................11

4. Safety................................................................................................................................... 12 4.1. Safety for the target species .....................................................................................................12

4.1.1. Tolerance studies .................................................................................................................12 4.1.2. Conclusion on safety for target species ...............................................................................13

4.2. Metabolism of astaxanthin.......................................................................................................13 4.3. Safety for the consumer – Studies on laboratory animals........................................................14

4.3.1. Acute toxicity ......................................................................................................................14 4.3.2. Genotoxicity, including mutagenicity .................................................................................14 4.3.3. Sub-chronic toxicity ............................................................................................................15 4.3.4. Chronic toxicity/Carcinogenicity ........................................................................................15

4.4. Reproduction toxicity, including teratogenicity ......................................................................17 4.5. Consumer safety ......................................................................................................................18

4.5.1. Margin of exposure..............................................................................................................18 4.5.2. Conclusion...........................................................................................................................19

4.6. Safety for the user ....................................................................................................................19 4.6.1. Effects on the respiratory system.........................................................................................19 4.6.2. Effect on eye and skin .........................................................................................................19 4.6.3. Sensitisation.........................................................................................................................19 4.6.4. Conclusion on user safety....................................................................................................19

4.7. Safety for the environment.......................................................................................................20 Conclusions and Recommendations............................................................................................ 20 Conclusions ................................................................................................................................. 20 Recommendations ....................................................................................................................... 21 Documentation provided to EFSA .............................................................................................. 22 References ................................................................................................................................... 22 Appendix ..................................................................................................................................... 24



BACKGROUND Regulation (EC) No 1831/20032 establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular, Article 4(1) of that Regulation lies down that any person seeking an authorisation for a feed additive or for a new use of a feed additive shall submit an application in accordance with Article 7.

The European Commission received a request from the company DSM Nutritional Products3 for authorisation of astaxanthin dimethyldisuccinate (CAROPHYLL® Stay-Pink) to be used as a feed additive for salmon and trout (category: sensory additive; functional group: colourants) under the conditions mentioned under 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). EFSA received directly from the applicant the technical dossier in support of this application. 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 16 of May of 2007.

The additive contains pure crystalline astaxanthin dimethyldisuccinate. The synthetic astaxanthin (E 161j) is authorised without a time limit at Community level for salmon, trout and ornamental fish.4 Astaxanthin-rich Phaffia rhodozyma (ATCC 74219) (E 161z) is authorised without a time limit for use in salmon and trout.5 Astaxanthin-rich Phaffia rhodozyma (ATCC SD-5340) (E 161y) is authorised for use in salmon and trout until 3.08.2011.6

The Scientific Committee on Animal Nutrition (SCAN) issued opinions on specific questions on the efficacy and safety of synthetic (EC, 1989) and biosynthetic [Phaffia rhodozyma (ATCC 74219)] (EC, 2002; EC, 2003) astaxanthin. The FEEDAP Panel has adopted three opinions on astaxanthin. One opinion on the environmental impact of astaxanthin-rich Phaffia rhodozyma (ATCC 74219) (EFSA, 2004); another one dealt with the safety of astaxanthin in animal nutrition (EFSA, 2005); in the third opinion the safety and efficacy of an astaxanthin-rich Phaffia rhodozyma (ATCC SD-5340) product were assessed (EFSA, 2006).

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 efficacy and the safety for the target animal(s), user and consumer and the environment, of astaxanthin dimethyldisuccinate (CAROPHYLL® Stay-Pink) when used under the conditions described in Table 1.

ACKNOWLEDGEMENTS The European Food Safety Authority wishes to thank the members of the Working Group on Colourants (Carophyll® Stay-Pink) for the preparation of this opinion.

2 OJ L 268, 18.10.2003, p.29 3 DSM Nutritional Products Sp.zo.o. Tarczynska 113. 96-320 Mszczonow. Poland 4 OJ L 298, 28.10.1998, p.4 5 OJ L 243 15.7.2004, p.10 6 OJ L 184 14.7.2007, p.12



Table 1. Register entry as proposed by the applicant

Additive Astaxanthin dimethyldisuccinate

Registration number/EC No/No (if appropriate) Not yet identified

Category of additive Sensory

Functional group of additive Colourants – Substances which, when fed to animals, add colours to food of animal origin

Description

Composition, description Chemical formula

Purity criteria (if appropriate)

Method of analysis (if appropriate)

Assay minimum of astaxanthin dimethyldisuccinate C50H64O10 Min. 96 % HPLC

Heavy metals Max. 10 ppm XRF Lead Max. 5 ppm XRF Mercury Max. 1 ppm XRF Arsenic Max. 2 ppm XRF Tryphenylphosphine oxide (TPPO) Max. 180 ppm GC Other carotenoids Max. 4 % HPLC Residue on ignition Max 0.1 % USP <281> 0.05 % solution in chloroform Clear Ph. Eur. <2.2.1> Absorption maximum wavelength (in chloroform) 484-493 nm Ph. Eur. <2.2.25>

Trade name (if appropriate) CAROPHYLL® Stay-Pink

Name of the holder of authorisation (if appropriate)

Conditions of use

Minimum content Maximum content Species or category of

animal Maximum

Age mg kg-1 of complete feedingstuffs

Withdrawal period

(if appropriate)

Salmonids (salmon and trout) - - - None

Other provisions and additional requirements for the labelling

Specific conditions or restrictions for use (if appropriate)

From 50 g of weight onwards. The combination of astaxanthin dimethyldisuccinate and canthaxanthin is allowed provided that the mixture does not exceed 138 mg/kg complete feedingstuffs

Specific conditions or restrictions for handling (if appropriate)

It is necessary to produce a stabilized form of astaxanthin dimethyldisuccinate for it to be marketed. Preparations of astaxanthin dimethyldisuccinate are aimed to be incorporate directly into the feed or through its incorporation in a premixture.

Post market monitoring (if appropriate) None

Specific conditions for use in complementary feedingstuffs (if appropriate)

None



Maximum Residue Limit (MRL) (if appropriate)

Marker residue Species or category of animal

Target tissue(s) or food products

Maximum content in tissues

Astaxanthin Salmon and trout Flesh 25 µg/g



ASSESSMENT

1. Introduction

CAROPHYLL® Stay-Pink (C®SP) is a feed additive, containing synthetic astaxanthin (ATX) as astaxanthin dimethyldisuccinate (ATX-dimethyldisuccinate). ATX-dimethyldisuccinate is an ester of ATX. It has been developed to overcome the potential loss of ATX faced during fish feed processing. The purpose of C®SP is to provide farmed Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) with a source of the carotenoid ATX that confers the characteristic pink colour to wild salmonids, which in nature is primarily obtained from crustaceans, a major part of their diet.

The applicant recommended a concentration of ATX-dimethyldisuccinate in complete feedingstuffs for fish in the range of 55 to 97 mg (corresponding to 40 to 70 mg ATX) kg-1 complete feedingstuff. The current maximum content authorised is 100 mg ATX ─together with canthaxantin─ kg-1 complete feed.

2. Characterisation of the product

ATX-dimethyldisuccinate is derived by esterification of ATX. Details of ATX-dimethyldisuccinate synthesis are provided in the dossier.7 C®SP is a dark reddish-brown crystalline powder. C®SP consists of 111 g ATX-dimethyldisuccinate, 20 g ethoxyquin, 11 g ascorbyl palmitate, 330 g porcine gelatine, 139 g sucrose, 139 g dextrin yellow and 250 g corn starch kg-1.8 1.38 g ATX-dimethyldisuccinate is equivalent to 1 g ATX; the product therefore contains 80 g ATX equivalents kg-1.

Total carotenoids in C®SP contain ≥96 % ATX-dimethyldisuccinate ((trans + 9-cis + 13-cis) isomers) including minor quantities of ATX monomethylsuccinate and free ATX. The other carotenoids (≤4 %) consist mainly of adonirubin methylsuccinate (<1.5%) and astacene dimethyldisuccinate plus semi-astacene dimethyldisuccinate (<1 %).9 ATX-dimethyldisuccinate (Figure 1) occurs mainly in the all-trans and also the 9-cis and 13-cis isomeric forms. It is present in three enantiomeric forms. NMR and mass spectra are given; ATX-dimethyldisuccinate maximum absorption wavelength is 484-493 nm (in chloroform).

Figure 1. All-trans astaxanthin dimethyldisuccinate (C5OH64O10)

2.1. Product identity10

Particle size analysis indicated that approximately 67 % of the particles were between 250 and 425 μm, none less than 150 μm. The bulk density of C®SP was found to be 0.58 kg L-1. 7 Technical Dossier. Section 2.2. 8 Technical Dossier. Additional documentation 9 Technical Dossier. Section 2.2. 10 Technical Dossier. Additional documentation



The dusting potential measured using the Heubach test was between 61-114 mg, indicating a low proportion of dust, on average 84 mg (for three batches).

Electrostatic properties of C®SP were found to be low, following measurements of quantity of product loss following multiple passages through a steel Agway funnel.

2.2. Purity

Analysis of residual solvents (five batches) showed a dichloromethane residue between 1160 and 8020 mg kg-1 active substance.11 This solvent is listed as Class II (solvents to be limited) in the EMEA Guideline VICH Topic GL18 with the maximum limit of 600 mg kg-1 veterinary medicinal products, active substances and excipients (EMEA, 2000).

Representative certificates of analysis (five batches) showed a maximum tryphenylphosphine oxide (TPPO) content of 30 mg kg-1 active substance.11 The Joint FAO/WHO Expert Committee on Food Additives (JECFA, 2006) stated in the specifications for food-grade synthetic lycopene and zeaxanthin that the contaminant TPPO did not raise safety concerns, and accepted a maximum concentration of 0.01 % TPPO in the above carotenoid preparations.

Since the analyses provided did not detect As, Pb and Hg, the maximum concentrations for those heavy metals given in the Register entry proposal appear rather high. The proposed maximum guaranteed levels correspond to the maximum permitted level (Directive 2002/32/EC)12 in complete feed for Pb and As, but exceed the authorised level for Hg by a factor of 10. Cd is not mentioned by the applicant.

2.3. Stability13

Stability studies with C®SP have been carried out to test the effects of various storage temperatures (15, 25 and 35 °C). Based on the ATX-dimethyldisuccinate content, the studies indicate a shelf-life of 24 months (losses <1 %) in closed bags. The stability of ATX-dimethyldisuccinate was reduced (approximately 2 % loss in one month, at storage temperatures of 25 or 35 °C) when stored in open bags.

Stability studies have also been performed with premixtures and complete feeds. Analysis of triplicate batches of premixtures indicates a minimal ATX-dimethyldisuccinate loss after the initial processing and three-month storage. The effect of feed processing and storage (for three months at 25 °C) on ATX-dimethyldisuccinate stability has been investigated in triplicate batches of complete feedingstuffs. Extrusion resulted in losses of approximately 10 % ATX-dimethyldisuccinate. During storage of processed feed, a monthly loss of approximately 2 % ATX-dimethyldisuccinate was observed in complete feedingstuffs.

One study in salmon (see Section 3.1) and four in trout (see Section 3.2) indicated monthly losses of about 3 % ATX from C®SP, independent of feed concentration (55 to 100 mg ATX equivalents kg-1 feed), which confirms the results obtained in the stability studies.

2.4. Homogeneity

Data were provided for premixtures and compound feeds containing C®SP to examine ATX-dimethyldisuccinate homogeneity. The analysis of ATX-dimethyldisuccinate in triplicate

11 Technical Dossier, Annex 2.7 12 OJ L140, 30.5.2002, p.10 13 Technical Dossier. Additional documentation



batches of premixtures and feed indicated satisfactory homogeneity with average coefficients of variation of 1.6 and 4.1 %, respectively.14

2.5. Evaluation of the analytical methods by the Community Reference Laboratory (CRL)

EFSA verified the report submitted by the Community Reference Laboratory (CRL) concerning the analytical method(s) for C®SP. The executive summary of the report is attached in the Appendix.

3. Efficacy

The applicant has provided a total of six trials with ATX-dimethyldisuccinate from C®SP in salmonids. C®SP has been tested at doses between 55 and 100 mg kg-1 ATX equivalents from C®SP. The trials were carried out on Atlantic salmon (one study) for nine months and on rainbow trout (five studies) for at least three months. One study in trout contained an unsupplemented control group without pigments. C®SP was compared to a corresponding amount of synthetic ATX (obtained from Carophyll® Pink (C®P)). Another study in trout was a dose-response trial. Efficacy was measured by the colouration of fish flesh (SalmoFanTM and redness ‘a’ value) and the concentration of ATX in flesh. In addition, the carotenoid deposition rate was calculated.

3.1. Efficacy trials in Atlantic salmon (Salmo salar L.)

C®SP was compared to synthetic ATX at a target concentration of 60 mg ATX equivalents kg-1 feed in 210 non-pigmented Atlantic salmon, each (triplicates, 480 g initial weight) for nine months (final weight 3400 g). ATX equivalents were analytically controlled during feed production and monthly for each batch (three batches, for three months each). ATX concentrations at the start of the experiment (analysed levels of synthetic ATX 59.2, C®SP 52.4 mg ATX equivalents kg-1 feed) were close to the target values. Both diets lost about 9 % (mean of three batches) of the initial ATX level. The fish were fed to excess in the morning. Water temperature in the tanks ranged from 6.5 to 12 °C. Mortality was very low, being in the range of one to three fish per tank.

No significant effect (p<0.05) of the diet was observed on specific growth rate or feed conversion. Fish flesh pigmentation was fairly similar for both diets (Table 2).

14 Technical Dossier. Additional documentation



Table 2. Pigmenting efficacy of astaxanthin dimethyldisuccinate from C®SP in Atlantic salmon. Duration of trial: nine months. Triplicate of 70 fish per treatment. Average values of 30 samples

Target concentration: 60 mg ATX

equivalents kg-1 feed

SalmoFanTM ‘a’ value (Hunterlab)

Astaxanthin in flesh (mg kg-1)

Astaxanthin deposition rate

(%) From C®SP 24.8a 24.6a ± 1.7 4.5a ± 0.9 5.5a ± 0.2 From synthetic ATX 24.9a 26.1b ± 1.3 5.0a ± 0.9 6.0a ± 0.2

a,b Different superscripts within a column indicate statistically significant differences (p<0.05)

3.2. Efficacy trials in trout (Oncorhynchus mykiss)

In the first trial, C®SP and synthetic ATX were given to trout (3x25 fish per group) for three months (initial weight 138 g, final weight 530 g) at a target concentration of 60 and 100 mg ATX kg-1 feed.15 The analysed ATX in the finished feed (63 and 104 mg per kg for C®P, 59 and 99 mg per kg for C®SP) corresponded to the target values. The ATX losses during 12 weeks amounted to 11 % in the C®P-60 group and to 9 % in the C®P-100 group, whereas no losses were found in both C®SP groups. Fish samples (15 fish per group) were taken after six and 12 weeks for redness and ATX determination in flesh. Results showed that the flesh ATX concentration was influenced by the dietary level, but not by the ATX source (Table 3). Redness values did not vary between the groups. Survival, growth and feed conversion showed no significant differences.

In the second trial, C®SP and synthetic ATX were given to trout (3x25 fish per group) for three months (initial weight 123 g, final weight 550 g) at a target concentration of 55 mg ATX kg-1 feed.16 The analysed ATX in the finished feed (49 mg per kg for synthetic ATX and C®SP) corresponded to the target value. The ATX losses during 12 weeks amounted to 9 % in both groups. Fish samples (15 fish per group) were taken after six and 12 weeks for redness and ATX determination in flesh. Colour measurements revealed no significant variations among the treatments. The ATX content in flesh did not significantly differ after six or 12 weeks (Table 3). No differences concerning zootechnical parameters were seen.

The third trial was conducted according to the design of the second trial (initial weight 132 g, final weight 551 g).17 The analysed ATX in the finished feed (51 mg kg-1 for synthetic ATX and 52 for C®SP) corresponded to the target value (55 mg kg-1). The losses over 12 weeks amounted to 14 % in the C®P-55 group and to 8 % in the C®SP-55 group. Fish samples (15 fish per group) were taken after six and 12 weeks for redness and ATX determination in flesh. The redness ‘a’, ATX concentration in the flesh and ATX deposition rate were not significantly different between the groups (Table 3).

In the fourth trial, lasting four months, a dose-response study was performed. Doses of 20, 40, 60 and 80 mg ATX kg-1 feed from synthetic ATX and the corresponding amounts from C®SP were supplemented (eight treatments, two or three replicates of 50 fish per replicate, initial bw 158 g, final bw approximately 690 g).18 Fish (five per replicate) were sampled after 4, 8, 12 and 16 weeks. The analysed ATX in the finished feed corresponded to the target values. The losses during eight weeks amounted to 5 % in the synthetic ATX group and to 8 % in the C®SP group. ATX deposition in flesh increased significantly with time of administration and dosage, while

15 Technical Dossier, Section 3.3., Study 1, Annex 3.1 16 Technical Dossier, Section 3.3., Study 3, Annex 3.3 17 Technical Dossier, Section 3.3., Study 4, Annex 3.4 18 Technical Dossier, Section 3.3., Study 5, Annex 3.5



no significant differences between the products C®SP and synthetic ATX were found. Concerning the dose response, results indicate that above 60 mg ATX equivalents kg-1 feed, the concentration of pigment in the flesh began to level off (Table 3).

A fifth trial on trout was not considered, because incorrect dosing of C®SP could not be excluded.19

Table 3. Pigmenting efficacy of astaxanthin dimethyldisuccinate from C®SP in trout

Target concentration in feed (mg ATX equivalents)

‘a’ value (Hunterlab)

Astaxanthin in flesh (mg kg-1)

Astaxanthin deposition rate (%)

Study 1:

60 15.4a ± 1.0 10.2a ± 0.5 15.3c ± 0.7 from C®SP

100 16.8a ± 0.4 12.1b ± 0.5 10.8a ± 0.5 60 16.5a ± 0.2 11.0a ± 0.8 16.9d ± 1.4 from synthetic

ATX 100 16.3a ± 1.1 12.7b ± 0.5 11.5b ± 0.5 Study 2:

from C®SP 55 12.2a± 0.8 8.8a± 0.1 16.7a ± 0.3from synthetic

ATX 55 12.5a ± 0.3 8.3a ± 0.7 16.3a ± 1.4

Study 3:

from C®SP 55 14.0a ± 0.4 8.8a ± 1.2 15.8a ±2.0 from synthetic

ATX 55 14.2a ± 2.0 8.4a ± 1.0 15.7a ± 2.2

Study 4:

20 10.7a ± 0.6 4.7a ± 0.4 20.2d ± 2.0 40 14.3b ± 0.4 8.0b ± 0.6 17.3c ± 1.2 60 15.3c ± 0.9 9.8c ± 1.2 14.4b ± 1.5

from C®SP

80 15.2c ± 0.7 10.1c ± 0.4 10.9a ± 0.7 20 10.3a ± 0.3 5.0a ± 0.3 22.1d ± 1.1 40 13.6b ± 0.2 8.6b ± 0.3 19.8d ± 0.8 60 15.6c ± 0.3 11.0c ± 0.8 17.0c ± 1.4

from synthetic ATX

80 16.1d ± 0.3 12.6d ± 0.9 14.1b ± 0.9 a,b,c,d: Different superscripts within a column and for a given study indicate statistically significant

differences (p<0.05)

3.3. Other effects on the quality of fish flesh

Effects on the organoleptic properties of fish flesh (e.g. taste) were not determined. However, they are unlikely to be affected, taking into account the history of ATX use in fish feeding.

3.4. Conclusions on efficacy of astaxanthin from CAROPHYLL® Stay-Pink

ATX-dimethyldisuccinate is efficacious in colouring the flesh of salmonids. This conclusion is based on studies designed to compare esterified with free ATX, which showed similar flesh redness and ATX content in the two groups, in salmon and trout. The efficacy was demonstrated in the dose range of 20 to 100 mg ATX equivalents kg-1 feed, redness with tissue deposition reaching a plateau at about 60 mg ATX equivalents kg-1 feed .

19 Technical Dossier, Section 3.3., Study 6, Annex 3.6



Differences in the ATX deposition rate between the esterified and non-esterified forms observed in two out of five trials were small but significant. Those differences, with inconsistent occurrence, are difficult to interpret.

4. Safety

4.1. Safety for the target species

Published literature data indicate the safety of ATX for the target species. In its opinions (i) on the safety of use of colouring agents in animal nutrition, Part I, General principles and Astaxanthin (EFSA, 2005), and (ii) on the safety and efficacy of an astaxanthin-rich Phaffia rhodozyma product (EFSA, 2006), the FEEDAP Panel concluded that “astaxanthin is tolerated by Atlantic salmon supplemented with dietary concentrations of about triple and by rainbow trout of about double the maximum content approved”. Higher tolerances could not be established because higher levels were not tested.

The former assessments did not include ATX-dimethyldisuccinate as the source of ATX. The applicant submitted one tolerance study with ATX-dimethyldisuccinate.

4.1.1. Tolerance studies

An eight-week feeding trial was conducted with 180 rainbow trout (Oncorhynchus mykiss) of 122 g initial body weight.20 Water temperature varied from 14.6 to 16.0 °C during the trial. Water quality was regularly monitored and kept below the critical levels of ammonia and nitrites.

Dietary treatments consisted of two control groups, a negative control with unsupplemented feed (C-0), and a positive control (C-100) with feed supplemented with 100 mg free ATX from synthetic ATX kg-1 feed. Two experimental groups received feed with 100 mg (E-100) and 1000 mg (E-1000) ATX equivalents (from ATX-dimethyldisuccinate) as C®SP. Each diet was fed to triplicate tanks (3 x 15 fish). Feed consumption decreased during the trial from 1.8 % initially to 1.3-1.4 % body weight at the end of the trial.

The extruded diets contained 40.6 % crude protein and 27.0 % total lipids (both analysed). Dietary ATX and ATX-dimethyldisuccinate were determined at the start and at the end of the trial. The initial levels confirmed the intended values (94.0 and 908 mg kg-1 for the groups C-100, E-100, and E-1000, respectively). The ATX losses by the end of the eight-week feeding period amounted to 11, 11, and 19 % for the groups C-100, E-100, and E-1000, respectively.

Survival, body weight, weight gain, feed conversion ratio and specific growth rate were measured and computed for each replicate. At the end of the feeding period, macroscopic observation was performed on ten individual fish per replicate tank (a total of 30 fish per treatment). Each fish was examined externally and then dissected for observation of viscera, liver, bile, spleen and muscle. Body weight, length, liver weight, condition factor and liver-somatic index were recorded on each sampled fish.

The administration of ATX-dimethyldisuccinate from C®SP at levels of 100 and 1000 mg ATX equivalents kg-1 did not result in any variation in performance as compared to both control groups (no fish died, specific growth rate= 1.82, feed:gain= 0.81). In all groups, the fish were healthy and in good nutritional condition (condition factor of 1.55, 1.56, 1.56, and 1.57 for the groups C-0, C-100, E-100, and E-1000, respectively); the organs were normal in all cases and no pathologic alterations were observed.

20 Technical Dossier. Section 4.1.1.



4.1.2. Conclusion on safety for target species

The tolerance study indicated that ATX-dimethyldisuccinate from C®SP was well tolerated at a dietary level of 908 mg ATX equivalents kg-1 complete diet. Due to the short duration of the trial (eight weeks), conclusions on the safety of ATX-dimethyldisuccinate from C®SP for the target animals are limited. However, as no evidence for concern was observed, and taking into account i) that ATX-dimethyldisuccinate is hydrolysed to free ATX and succinate before or during intestinal absorption (see Section 4.2.1.), ii) that succinate is an endogenous metabolite, and iii) the former assessments of ATX (EFSA, 2005; EFSA, 2006), the FEEDAP Panel considers ATX-dimethyldisuccinate from C®SP safe for the target species.

4.2. Metabolism of astaxanthin

4.2.1. Metabolic fate of astaxanthin dimethyldisuccinate

A study of the comparative metabolism of ATX and ATX-dimethyldisuccinate has been carried out in rainbow trout (Oncorhynchus mykiss).21 Two groups of six animals each received 40 mg ATX from synthetic ATX (group 1) and 40 mg ATX equivalents from ATX-dimethyldisuccinate from C®SP (group 2). After one-week supplementation, fish received a single capsule containing either [6,7,6’,7’-14C]-ATX (group 1) or [6,7,6’,7’-14C]-ATX-dimethyldisuccinate (group 2) to a dose equivalent to 100 mg kg-1 feed. One or two animals were sacrificed one-day post-dosage, and the rest of the fish after four days. Faeces were recovered by stripping at sacrifice.

The highest levels of radioactivity were measured in the blood, liver, kidney and bile after one day when compared to day 4. Those levels increased in muscle, skin and fat at least until day 4. Blood radioactivity levels after one day were equivalent to 1.8 and 3.1 mg equivalents kg-1 for groups 1 and 2, respectively. Muscle radioactivity was also higher in the ATX-dimethyldisuccinate group when compared to the ATX (0.10 and 0.09 mg equivalent ATX kg-1 after day 1, 0.27 and 0.11 after day 4).

Analysis of the gastrointestinal contents showed that ATX-dimethyldisuccinate was already hydrolysed extensively in the stomach, and that only 10 % remained unchanged in the intestine at one-day post-dosage. About 92 % of the plasma radioactivity corresponded to ATX isomers, mainly all-E with minor 9-Z and 13-Z; no free ATX-dimethyldisuccinate or ATX methylsuccinate were detected in plasma, even after one-week feeding. Also in the liver, only isomers of ATX, but not of ATX-dimethyldisuccinate, could be detected. From the extracted radioactivity in muscle, ≥80 % corresponded to free ATX (apolar extracts) and ≤12 % to polar extracts consisting of at least eight metabolites.

4.2.2. Published data

In its opinion on the use of ATX in animal nutrition (EFSA, 2005), the FEEDAP Panel described the metabolism of ATX in detail. The main features are summarised without the corresponding literature references.

ATX apparent absorption is determined by several factors such as: i) fish species and strains, ii) ATX form, ATX esters being less bioavailable than free ATX and the geometrical isomer all-E being absorbed more efficiently than the Z isomers, whereas no difference was observed for the 3S,3’S, 3R,3’R or 3R,3’S meso enantiomers, and iii) dietary factors such as lipid levels. The apparent absorption varies from 20 to 95 %, with most values comprised between 50 and 70 %.

21 Technical Dossier, Section 4.1.3.1., Annex 4.4.



ATX is metabolised in fish mainly through reductive pathways. A double-step reduction at the 4 and 4’-oxo groups initiates a metabolic process leading to idoxanthin and/then to adonixanthin and finally zeaxanthin. No oxidation occurs in ‘sea bream type’ of fish like salmonids and therefore the conversion of zeaxanthin to ATX does not occur.

ATX has been shown to be a vitamin A precursor for fish, which implies the cleavage of the polyene chain.

After ATX administration, the pigment deposited in the flesh of trout and Chinook salmon is predominantly ATX (about 95 %); in arctic charr (Salvelinus alpinus), also idoxanthin is deposited (20-35 %). A dose-related increase of ATX in the flesh of trout and salmon was observed according to the ATX levels in the diet, a plateau being reached in salmon. The composition of carotenoids deposited in the flesh reflects that of the dietary pray organisms or added carotenoids in term of ATX stereo isomers. All-E isomers are deposited mainly in flesh whereas Z isomers are preferentially stored in the liver and kidney.

4.2.3. Conclusions

ATX-dimethyldisuccinate is hydrolysed and converted extensively to free ATX in the intestine of fish, then absorbed, metabolised and distributed in the same manner as free ATX.

4.3. Safety for the consumer – Studies on laboratory animals

4.3.1. Acute toxicity

Acute oral toxicity for synthetic ATX in mice and rats was very low (>8000 mg kg-1) as was for ATX-dimethyldisuccinate from C®SP in female rats (>2000 mg kg-1).22

4.3.2. Genotoxicity, including mutagenicity

ATX-dimethyldisuccinate as C®SP was tested in a bacterial reverse mutation assay using the standard Salmonella typhymurium strains TA 97a, 98, 100, 102 and 1535 (Ames test).23 Both a plate incorporation test and a preincubation test were conducted, with and without metabolic activation. No increase in revertants (gene mutations) was observed at concentrations up to 2500 µg (active ingredient) plate-1.

A chromosome aberration test was conducted with ATX-dimethyldisuccinate as C®SP in human lymphocytes in vitro, with and without metabolic activation, according to OECD guideline 473.24 Concentrations were up to precipitation, which occurred at lower concentrations when tested without S9 metabolic activation. Maximum concentration achieved without precipitation was 1000 µg C®SP mL-1, corresponding to 160 µg ATX-dimethyldisuccinate mL-1. No significant increase in chromosome aberration was observed.

A micronucleus test in rat bone marrow cells was conducted according to OECD guideline 474 at doses up to 667 mg ATX-dimethyldisuccinate kg-1 bw as C®SP by gavage (twice).25 No statistically significant increase in micronuclei was reported.

In conclusion, two in vitro and one in vivo assays demonstrate the absence of genotoxic potential of the additive C®SP.

22 Technical Dossier, Annex 4.6 23 Technical Dossier, Annex 4.8 24 Technical Dossier, Annex 4.9 25 Technical Dossier, Annex 4.10



4.3.3. Sub-chronic toxicity

4.3.3.1. Rats

A 28-day range finding toxicity study (OECD guideline 407) was conducted in SPF Wistar rats at doses of 100, 500 and 1000 mg ATX-dimethyldisuccinate from C®SP kg-1 bw day-1.26 Parameters were clinical signs, food consumption, body weight, haematology, clinical chemistry, gross and histopathology. No mortality occurred. Faeces were coloured and soft in the mid- and high-dose groups near the end of the study, which was attributed to the properties of the colourant. Plasma determination indicated adequate uptake of the additive and no other effects were observed in any of the endpoints measured. The NOAEL from this study was >1000 mg kg-1 bw day-1.

A 13-week oral toxicity study (OECD guideline 408) was conducted in SPF Wistar rats.27 Doses were 50, 150 or 500 mg ATX equivalents as ATX-dimethyldisuccinate from C®SP kg-1 bw day-1. Control groups (untreated and placebo) were included as well as satellite groups for the assessment of reversibility. No clinical abnormalities were found except for red-coloured faeces in the treated groups. No abnormalities were found in functional and ophthalmological tests. In clinical chemistry, an increase in cholesterol was seen in both sexes in the highest dose group, as well as serum sodium, potassium and calcium concentrations. Sodium was also increased (water consumption not given) in the 150 and 50 (females only) dose groups. Serum LDH was increased in 500 mg kg-1, and females had increased creatinine kinase. Bilirubin was increased in the 150 and 500 mg kg-1 female. These effects appeared to be reversible after four weeks. Gross pathology showed orange colouration of adipose tissue of all exposed rats, including those of the recovery group. In histopathology slight to minimal hypertrophy of the adrenal cortex was noted in some animals of the high-dose group and one of the mid-dose group. Based on these findings, the FEEDAP Panel identified a NOAEL of 50 mg ATX equivalents kg bw-1 day-1.

An earlier 13-week toxicity study of synthetic ATX in rats at doses from 310 to 1240 mg kg-1 bw day-1 showed a similar range of effects to those described above but failed to provide a NOAEL.

4.3.3.2. Dogs

A 13-week study was performed with a 6 % synthetic ATX preparation where three Beagle dogs per group and sex were fed doses equivalent to 0, 40, 80 and 160 mg synthetic ATX kg-1 bw day-1.28 The study showed no treatment-related effect on clinical signs, body weight, feed intake, haematology, clinical chemistry, urinalysis, ophtalmoscopy, gross- and histopathology (except for yellow colouration of adipose tissue seen in the ATX groups).

4.3.4. Chronic toxicity/Carcinogenicity

4.3.4.1. Rats

A 52-week study was conducted in SPF Wistar rats using an 8 % synthetic ATX preparation (water miscible food grade).29 Intended doses of 0, 125, 250, 500 and 1000 mg ATX kg-1 bw day-1 were given via feed. Satellite groups were included for reversibility study (51 weeks recovery). Parameters included in the study were clinical observations and examination 26 Technical Dossier, Annex 4.11 27 Technical Dossier, Annex 4.12 28 Technical Dossier, Annex 4.14 29 Technical Dossier, Annex 4.15



(mortality, body weight, food and water consumption, ophtalmoscopy, haematology, clinical chemistry, urinalysis), necropsy including recording of organ weights, and histopathology. Faecal discolouration predominantly in the higher dose groups was the only clinical abnormality recorded. Decrease in weight gain was seen in animals treated with ATX and placebo, but in females there was also a difference between placebo control and animals dosed at 250 mg and higher. Absolute food consumption decreased in test and placebo control groups but relative food consumption was reduced compared to the placebo group. Haematology values revealed slight variations compared to those of the placebo control, but without dose response and within the physiological range. Serum cholesterol was increased in both sexes at 125 mg and higher compared to the placebo control. Bilirubin was increased in males (from 500 mg) and females (from 125 mg). ALAT and ALP levels were increased in females (from 250 mg) as was ASAT (from 500 mg). Males had lower ALAT levels (1000 mg). Urinalysis revealed increased specific gravity, notably in females, from 250 mg onwards. Relative organ weights were decreased, such as spleen, adrenals, ovaries, liver, brain and kidney. These changes were most pronounced in females (except kidney) and occurred already at lower doses (125-250 mg). Histopathological changes were confined to the liver and were more pronounced in females. These included brownish pigmentation of hepatocytes and macrophages in all dose groups; the pigment was not identified. Centrolobular hypertrophy was seen in females from 250 mg onwards, and inflammatory cell foci and multinucleated hepatocytes were significantly increased in the high-dose females. From this study, the FEEDAP Panel cannot establish a NOAEL.

The same preparation was tested for carcinogenicity in Wistar rats, 60 animals per dose and sex, 104 weeks duration and doses 0, 0 + carrier, 40, 200 or 1000 mg kg-1 bw day-1.30 A subgroup was allowed to recover after one year till the end of the study. Clinical signs included red colouration of faeces in the higher dose groups. Mortality was relatively high in the controls, in particular females (56 % survival). Body weight gain was reduced compared to the carrier control in the 200 and 1000 mg female groups; this was compensated for during the recovery period. In haematology slight changes were observed in the higher dose groups. In clinical chemistry, slight changes were seen, mainly in females in the 1000 mg group and, occasionally, in the 200 mg group. These included increase in cholesterol, bilirubin, ALAT and ASAT. At necropsy no differences among the groups were seen, except for the expected colouration of the adipose tissues. Relative organ weights of kidney and testis were decreased in the 1000 mg group males, and decreased ovary and adrenal weight in the 200 and 1000 mg group females. These organ changes were not associated with histopathological alterations. Histopathology at the end of the study showed exclusively treatment related changes in the liver such as centrolobular vacuolization in the 200 and 1000 mg male groups. In females more extensive changes were seen, indicating hepatocellular damage, regeneration and adaptation. These occurred mainly in the 1000 and 200 mg groups, but mild effects were also observed in the 40 mg group such as hepatocellular and focal vacuolization, fatty change and multinucleate and hypertrophic hepatocytes. Treatment related neoplastic changes were also confined to the liver: hepato-cellular adenomas were increased in the 200 and 1000 mg groups of females only, while in the low dose group the increase was not statistically significant. In view of the neoplastic changes in the liver of females these changes are considered secondary to liver damage and regeneration.

The FEEDAP Panel therefore concludes that the benign liver tumours observed in the study, together with the absence of genotoxicity, do not indicate a carcinogenic potential and consequently, setting a NOAEL would be possible. However, because mild effects were

30 Technical Dossier, Annex 4.17



observed in the lowest dose group, the NOAEL is below 40 mg ATX (from a synthetic ATX preparation) kg-1 bw day-1.

4.3.4.2. Mice

A 80-week carcinogenicity study was conducted in NMRI MORO (SPF) mice with a preparation of synthetic 8 % ATX in the diet at concentrations of 0, 0+carrier, 14, 300, 650 or 1400 mg ATX kg-1 bw day-1.31 Mortality was relatively high (38-50 %) without differences among the groups. The only treatment-related clinical abnormality was (as expected) fecal discolouration. During the last six months, there was a decrease in body weight in the 300 mg and higher groups. In laboratory investigations, cholesterol was increased in the 1400 mg group. Necropsy showed no treatment-related differences in non-neoplastic or neoplastic lesions (confirmed by histopathology), only discolouration of adipose tissue in some animals from the higher dose groups.

The NOAEL from this study is 14 mg ATX kg-1 bw day-1 although the interval with the LOAEL (300 ATX kg-1 bw day-1) is relatively large.

4.3.4.3. Dogs

In a 52-week chronic toxicity study, four Beagle dogs per sex and group were given daily for 371 days gelatin capsules containing 0, 6, 24 and 96 (from the sixth month onwards, 200) mg ATX kg-1 bw from an 8 % preparation of synthetic ATX.32 No effects were seen in the parameters evaluated (clinical signs, body weight, feed intake, haematology, clinical chemistry, urinalysis, organ weight, gross- and histopathology) that could be related to the application of ATX, except for colouration of fatty tissue. The NOAEL from this study is considered to be 200 mg kg-1 day-1, based on the highest dose, longest duration and time of measurement of endpoints.

4.4. Reproduction toxicity, including teratogenicity

Synthetic ATX was given daily by gavage to male and female rats (32 per group) at doses of 0, 25, 100 or 400 mg kg-1 bw at appropriate periods in relation to mating.33 Males were dosed for 70 days prior to mating until sacrifice and females for 14 days prior to mating until sacrifice or until weaning; the F1 was not further treated. Half of the females were killed at gestation (day 14) for uterus examination. Remaining litters were examined for development, functional tests and reproductive capability. In the P generation, no treatment-related effects were seen on mating behaviour and uterine inspection. No effect from ATX exposure was seen in the observations made on F1 and F2 pups. Therefore, the NOAEL in this study is 400 mg ATX kg-

1 bw day-1 (the highest dose tested).

A two-generation study was conducted in rats using 0, 100, 250 or 800 mg synthetic ATX kg-1 bw day-1 in their diets for ten weeks prior to mating.34 F1 was subject to necropsy or allowed to mate for the F2 generation. Parameters recorded were clinical condition, body weight, food consumption, reproductive performance parameters, development and gross pathology. The results showed colouring effects of adipose tissue, gastrointestinal content, faeces and fur. Toxicity signs were found in the 800 mg kg-1 group (P and F1), seen as retardation of growth during pregnancy and retardation of pup growth during lactation and post weaning. In the 250

31 Technical Dossier, Annex 4.18 32 Technical Dossier, Annex 4.16 33 Technical Dossier, Annex 4.19 34 Technical Dossier, Annex 4.20



mg group there was slight retardation of F1 pups in the postweaning period. The NOAEL from this study was 100 mg kg-1 bw day-1.

Synthetic ATX was tested for embryotoxicity/teratogenicity in rats at doses of 0, 250, 500 or 1000 mg ATX kg-1 bw day-1 during gestation, day 7 to 16.35 One subgroup was necropsied on gestation day 21 and one subgroup on postnatal day 23. Parameters measured were body weight, gross pathology (notably female reproductive tract), and morphology of embryos and offsprings. No adverse effects were seen except for a dose-related reduction in body weight in the P generation. Examination of embryos did not reveal signs of embryotoxicity or teratogenicity. Therefore, the NOAEL in this study is 1000 mg ATX kg-1 bw day-1 (the highest dose tested).

Embryotoxicity/teratogenicity was tested in rabbits administered 0, 100, 200 or 400 mg synthetic ATX kg-1 bw day-1 by gavage during gestation, day 7 to 16.36 On day 30, the dams were killed, reproductive tract was examined for implantations, resorptions and corpora lutea, and the fetuses for visceral and skeletal abnormalities. No adverse effects were seen on the parameters studied in dams and fetuses. Therefore, the NOAEL in this study is 400 mg ATX kg-1 bw day-1 (the highest dose tested).

4.5. Consumer safety

The FEEDAP Panel recognises that many of the toxicity studies (e.g. chronic/carcinogenic and reproduction/teratogenicity) have been done in the early 90’s with synthetic ATX and not with ATX-dimethyldisuccinate. However, given the known metabolism of ATX-dimethyldisuccinate, and its hydrolysis in the digestive tract, the Panel considers that the studies are relevant to the safety assessment of C®SP.

Four subchronic (three in rats and one in dogs), and four chronic (two in rats, one in mice and one in dogs) toxicity studies with ATX were assessed. A lowest NOAEL was found in the mice carcinogenicity study with 14 mg ATX kg-1 bw day-1 (based on a decline in bw). No treatment-related differences in non-neoplastic or neoplastic lesions could be found in the mice study up to 1400 mg ATX kg-1 bw day-1. However, a NOAEL for ATX could not be established in the rat carcinogenicity study. Treatment-related liver changes (centrolobular vacuolisation in males, hepatocellular damage, regeneration and hepatocellular adenomas in females) were seen at 200 and 1000 mg ATX kg-1 bw day-1; in the group with 40 mg ATX kg-1 bw day-1, the increase in liver alterations was not statistically significant. Considering the absence of genotoxicity in ATX, the FEEDAP Panel concludes that these findings do not indicate a carcinogenic potential. The neoplastic changes in the liver are considered secondary to liver damage and regeneration.

4.5.1. Margin of exposure

A margin of exposure can be derived, according to the scenario described in a previous opinion of the FEEDAP Panel (EFSA, 2007), based on high intake of 165 g (fish and seafood) day-1

adult person-1, and on production figures for salmon and trout resulting in the consumption of

110 g salmon and 55 g trout. Considering the plateau levels of ATX deposition in salmon (10 mg kg-1) and trout (25 mg kg-1) (EFSA, 2005), a consumer exposure of 2.5 mg ATX (1.10 mg from salmon and 1.375 from trout) day-1 would result, which is a factor of 960 lower than the

35 Technical Dossier, Annex 4.21 36 Technical Dossier, Annex 4.22



LOAEL from the carcinogenicity rat study, and 336 times lower than the NOAEL from the mouse carcinogenicity study.

4.5.2. Conclusion

The FEEDAP Panel concluded in 2005 that ‘Supplementing salmonid feed with astaxanthin would not increase flesh astaxanthin of farmed fish essentially compared to wild catches. The FEEDAP Panel considers therefore the use of astaxanthin as feed additive to salmonid feed at the maximum level approved safe for the human consumer. This is in agreement with current FDA regulations.’ (EFSA, 2005)

The data submitted in the C®SP dossier indicated particularly that the colourant was neither genotoxic (ATX-dimethyldisuccinate) nor carcinogenic (ATX), and does not give rise for concern. Consequently, supplementation of fish feed with ATX at the highest approved level (100 mg kg-1 feed) is unlikely to represent an additional risk to the consumer.

Given the nature of ATX-dimethyldisuccinate, its breakdown in the alimentary tract to ATX and its deposition as ATX in flesh, the FEEDAP Panel considers that no new or additional safety concerns from the use of C®SP (ATX-dimethyldisuccinate), a substitutive product of other ATX sources, would originate for consumers.

4.6. Safety for the user

4.6.1. Effects on the respiratory system

No inhalatory toxicity study is provided.

4.6.2. Effect on eye and skin

ATX-dimethyldisuccinate (0.1 g) was instilled in one eye of three rabbits and evaluated up to 72 hours thereafter.37 Slight to moderate cornea and sclera irritation was observed, which had disappeared near the end of the observation period. Therefore the compound is classified as ‘non-irritant for the eyes’.

A skin irritation study was conducted in three albino rabbits.38 For four hours, 0.5 g of ATX-dimethyldisuccinate was applied to the skin. Due to the staining properties of the test compound, reddening could not be assessed shortly after application. However, after 24 hours and thereafter the skin did not show any reaction to the test compound and was therefore classified as ‘non-irritant for the skin’.

4.6.3. Sensitisation

Skin sensitisation was tested in a local lymph node assay, with ATX-dimethyldisuccinate in dimethylformamide up to 10 %.39 The stimulation index was below the trigger value, ATX-dimethyldisuccinate is therefore not classified as a skin sensitiser.

4.6.4. Conclusion on user safety

No dermal or ocular risk for the users of C®SP is likely to occur under practical conditions. In the absence of an acute inhalation study, the acute inhalatory toxicity cannot be established.

37 Technical Dossier, Annex 4.31 38 Technical Dossier, Annex 4.30 39 Technical Dossier, Annex 4.32



However, since the acute oral toxicity is very low (>2000 mg kg-1 bw) and particle size is >150 µm, systemic exposure through the respiratory tract is considered of limited risk.

4.7. Safety for the environment

Since ATX-dimethyldisuccinate is metabolised to ATX in fish, and excreted mainly in this form, the potential environmental risks are predominantly from ATX. In its opinion on the safety of use of colouring agents in animal nutrition (EFSA, 2005), the FEEDAP Panel concluded that: ‘No data are available for a qualified assessment on the environmental impact of astaxanthin in salmonid feed. Astaxanthin occurs naturally in the habitat of wild living salmonids. Astaxanthin as feed additive for farmed fish substitutes natural sources. As astaxanthin is insoluble in water and susceptible to oxido-reduction it will mainly bind to faeces and sink to the seabed. Idoxanthin and zeaxanthin, the main metabolic excretion products of astaxanthin, occur frequently and in considerable quantities in the environment. In that respect the FEEDAP Panel does not expect that the use of astaxanthin as feed additive to salmon and trout will pose a significant risk to the environment.’

The FEEDAP Panel does not expect that the use of C®SP containing ATX-dimethyldisuccinate will pose a risk to the environment.

CONCLUSIONS AND RECOMMENDATIONS

CONCLUSIONS

Orally administrated ATX-dimethyldisuccinate from C®SP is hydrolysed and converted to free ATX in the intestine of fish, then absorbed, metabolised and distributed in the same manner as free ATX.

ATX-dimethyldisuccinate from C®SP is efficacious in colouring the flesh of salmonids. This conclusion is based on studies comparing ATX-dimethyldisuccinate with free synthetic ATX, which produced similar results concerning flesh redness and ATX content in salmon and trout. Efficacy was demonstrated in the dose range of 20 to 100 mg ATX equivalents kg-1 feed, redness with tissue deposition reaching a plateau at about 60 mg ATX equivalents kg-1 feed .

A dietary level of 908 mg ATX equivalents kg-1 complete diet provided as ATX-dimethyldisuccinate from C®SP was well tolerated in rainbow trout for eight weeks but the short duration of the trial allows only limited conclusions. Taking into account former assessments of ATX, the FEEDAP Panel considers ATX-dimethyldisuccinate from C®SP, at the highest authorised ATX level in feed, safe for the target species.

The data submitted in the C®SP dossier, indicated particularly that the colourant was not genotoxic (ATX-dimethyldisuccinate), carcinogenic or teratogenic (ATX). Consequently, supplementation of fish feed with ATX at the highest approved level (100 mg kg-1 feed) is unlikely to represent an additional risk to the consumer. Given the nature of ATX-dimethyldisuccinate, its breakdown in the alimentary tract to ATX and its deposition as ATX in flesh, the FEEDAP Panel considers that no new or additional safety concerns from the use of C®SP (ATX-dimethyldisuccinate), a substitutive product of other ATX sources, would originate for consumers.

No dermal or ocular risk for the users of C®SP is likely to occur under practical conditions. In the absence of an acute inhalation study, the acute inhalatory toxicity cannot be established. However, since the acute oral toxicity is very low (>2000 mg kg-1 bw) and particle size is >150 µm, systemic exposure through the respiratory tract is considered of limited risk.



The FEEDAP Panel does not expect that the use of C®SP containing ATX-dimethyldisuccinate will pose a risk to the environment.

RECOMMENDATIONS

MODIFICATIONS TO THE REGISTER ENTRY

1. Description

The FEEDAP Panel recommends the modification of the section ‘Description’, particularly by introducing the content of ATX-dimethyldisuccinate and ATX equivalents of C®SP, a maximum content of dichloromethane and a reduction of the maximum TPPO content, as follows:

Description

Composition, description Chemical formula

Purity criteria (if appropriate)

Method of analysis (if appropriate)

Astaxanthin dimethyldisuccinate C50H64O10 Min. 111 g kg-1 HPLC - Astaxanthin equivalent Min. 80 g kg-1

Total carotenoids content - Astaxanthin dimethyldisuccinate Min. 96 % HPLC - Other carotenoids Max. 4 % HPLC

Heavy metals Max. XRF Lead Max. XRF Mercury Max. XRF Arsenic Max. XRF Cadmium Max. XRF Tryphenylphosphine oxide (TPPO) Max. 100 ppm GC Dichloromethane Max. 600 ppm GC

Undesirable substances should be avoided wherever possible. The applicant should therefore adjust the maximum guaranteed levels to the analysed data for Pb, Hg, As and Cd. The FEEDAP Panel cannot propose values because the LOQ and the LOD for the method applied were not given.

Considering the maximum accepted TPPO content in the JECFA specifications for synthetic lycopene and zeaxanthin (max. 100 mg kg-1), and the fact that the applicant has provided five certificates of analysis showing TPPO values (≤30 mg kg-1) considerably lower than the proposed maximum guaranteed level (180 mg kg-1), the FEEDAP Panel recommends to lower the TPPO content of the additive to a maximum value of 100 mg kg-1.

As a solvent residue, a maximum limit of 600 mg dichloromethane kg-1 product (veterinary medicinal products, active substances and excipients) has been set by EMEA (Guideline VICH Topic GL18). This maximum limit should also be applied to feed additives. Therefore, the FEEDAP Panel recommends a guaranteed maximum level of 600 mg dichloromethane kg-1 additive.

2. Conditions of use

According to the existing regulations on the maximum content of ATX (100 mg kg-1 complete feed), the maximum content of ATX-dimethyldisuccinate should be given as 138 mg kg-1 complete feed.

3. Specific conditions or restrictions for use



To be in line with current authorisations of ATX, the use of C®SP should be restricted from the age of six months onwards (instead of from 50 g of weight onwards, as proposed by the applicant).

The combination of ATX-dimethyldisuccinate and canthaxanthin should be allowed provided that the sum does not exceed 100 mg from ATX equivalents plus canthaxanthin kg-1 complete feedingstuffs for fish (instead of 138 mg ATX-dimethyldisuccinate, as proposed by the applicant).

4. Maximum Residue Limit (MRL)

The FEEDAP Panel does not see a necessity to introduce MRLs for ATX.

OTHER RECOMMENDATIONS

The ethoxyquin content of the product (20 g kg-1) should be labelled to help the feed compounder not to exceed the maximum content authorised in complete fish feed.

DOCUMENTATION PROVIDED TO EFSA 1. Astaxanthin dimethyldisuccinate (a sensory additive in feed for salmonids). October 2006.

Submitted by DSM Nutritional Products.

2. Additional documentation for the application for astaxanthin dimethyldisuccinate as a Sensory Additive for use in feed for salmonids (salmon and trout). April 2007. Submitted by DSM Nutritional Products.

3. Evaluation report of the Community Reference Laboratory feed additives authorisation on the methods(s) of analysis for Astaxanthin dimethyldisuccinate. August 2007.

4. Comments from the Member States received through the EFSAnet.

REFERENCES EC (European Commission). 1989. Report of the Scientific Committee for Animal Nutrition on

the use of Astaxanthin in feedingstuffs for salmon and trout. <http://ec.europa.eu/food/fs/sc/oldcomm6/other/01_en.pdf>

EC (European Commission). 2002. Report of the Scientific Committee for Animal Nutrition on the use of Astaxanthin-rich Phaffia rhodozyma in feedingstuffs for salmon and trout. <http://ec.europa.eu/food/fs/sc/scan/out76_en.pdf>

EC (European Commission). 2003. Update of the opinion of the Scientific Committee on Animal Nutrition on the use of Astaxanthin-rich Phaffia rhodozyma in feedingstuffs for salmon and trout. <http://ec.europa.eu/food/fs/sc/scan/out111_en.pdf>

EFSA (European Food Safety Authority). 2004. Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed on environmental impact of Astaxanthin-rich Phaffia rhodozyma (Ecotone®) as feed additive in accordance with Council Directive 70/524/EEC. <http://www.efsa.eu.int/science/feedap/feedap_opinions/366_en.html>

EFSA (European Food Safety Authority). 2005. Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed on the safety of use of colouring agents in animal nutrition. PART I. General Principles and Astaxanthin. <http://www.efsa.eu.int/science/feedap/feedap_opinions/1263_en.html>



EFSA (European Food Safety Authority). 2006. Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed on a request from the European Commission on the safety and efficacy of the product AQUASTA, an Astaxanthin-rich Phaffia rhodozyma ATCC SD-5340 for salmon and trout. <http://www.efsa.europa.eu/en/science/feedap/feedap_opinions/1369.html>

EFSA (European Food Safety Authority). 2007. Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed on the Maximum Residue Limits of canthaxanthin coming from animals fed with canthaxanthin used as a feed additive in accordance with Council Directive 70/524/EEC. <http://www.efsa.europa.eu/en/science/feedap/feedap_opinions/ej507_canthaxanthin.html>

EMEA (European Medicines Agency), 2000. Impurities: Residual Solvents in new veterinary medicinal products, active substances and excipients. VICH topic GL18, Step 7. <http://www.emea.europa.eu/pdfs/vet/vich/050299en.pdf>

JECFA (Joint FAO/WHO Expert Committee on Food Additives). 2006. Compendium of food additive specifications. 67th meeting. <ftp://ftp.fao.org/docrep/fao/009/a0675e/a0675e00.pdf>



APPENDIX Executive Summary of the Evaluation Report of the Community Reference Laboratory Feed Additives Authorisation on the Method(s) of Analysis for CAROPHYLL®Stay Pink

Astaxanthin dimethyldisuccinate (AXN-DMDS) is a product for which authorisation is sought as feed additive under the category "sensory additives", functional group "colorants: substances which, when fed to animals, adds colours to food of animal origin", according to the classification system of Annex I of Regulation (EC) No 1831/2003. The active substance is all-trans astaxanthin dimethyldisuccinate which is a specific geometrical isomer of AXN-DMDS. The applicant intends to place the product on the market in the specific beadlets formulation Carophyll® Stay Pink.

The product contains in addition to all-trans AXN-DMDS about 1.5 % of two cis isomers of AXN-DMDS and less than 4 % of carotenoids other than AXN-DMDS. It is intended to add the feed additive to fish feed for salmon and trout at a recommended rate ranging from 55 to 97 mg/kg of the active substance in complete feedingstuff. The recommended maximum concentration of the active substance is 138 mg/kg of complete feedingstuff. The concentration of the active substance in all matrices is measured as the sum of the concentration of all-trans and two cis isomers of AXN-DMDS.

The applicant proposes analytical methods for the determination of the active substance that are specifically designed for this particular product formulation (beadlets). The analytical procedures for the analysis of AXN-DMDS in the formulated product, premixtures and feedingstuffs are similar and are comprised of the following steps: (1) The enzymatic release of AXN-DMDS from the beadlets formulation, (2) the extraction of AXN-DMDS with dichloromethane and ethanol, (3) the purification of the extract with solid phase extraction columns when analysing feedingstuffs and (4) the determination of AXN-DMDS by normal phase high performance liquid chromatography (HPLC) coupled to ultraviolet detection (UV detection) measuring at about 470 nm. The method allows for the simultaneous determination of the all-trans and cis isomers of AXN-DMDS and the other carotenoids present in the matrix, since these substances are well separated in the HPLC chromatogram.

Method performance characteristics were determined on feedingstuffs containing AXN-DMDS of about 50 mg/kg, obtaining a percentage recovery rate of 98 % and a relative within laboratory reproducibility standard deviation of about 2%. The limit of quantification was 0.2 mg/kg. Performance characteristics for other carotenoids have not been provided. Based on the results from the validation study the method is considered suitable for official control purposes to determine the active substance in feedingstuffs within the frame of this application.

The applicant proposed a Maximum Residue Limit (MRL) for astaxanthin in the target fish tissue of 25 mg/kg, for which an analytical method has been proposed. The astaxanthin content is expressed in terms of the concentration of the sum of the measured geometrical isomers of this compound. The method is comprised of two steps, which are the extraction of the sample with acetone and the determination of the target analytes with HPLC coupled to UV detection. The parameters of the HPLC detection of the target analytes in all matrices (i.e fish tissue, formulations, premixtures and feedingstuffs) are identical.

Method performance characteristics were determined on fish flesh fortified with astaxanthin. The percentage recovery rate was 98 % obtained on samples containing 10 mg/kg of the target analyte and the relative within laboratory reproducibility standard deviation was about 3 %, obtained on samples containing about 6.5 mg/kg of the target analyte. The limit of



quantification was 0.2 mg/kg. Based on the results from the validation study the method is considered suitable for official control purposes.

The applicant proposed limits of various impurities in the feed additive including some heavy metals for which appropriate methods are available.

Further testing or validation by the CRL is not considered necessary.

astaxanthin dimethyldisuccinate

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